Naoto Endo

Maturational disturbance of chondrocytes in Cbfa1-deficient mice.

Cbfa1, a transcription factor that belongs to the runt‐domain gene family, plays an essential role in osteogenesis. Cbfa1‐deficient mice completely lacked both intramembranous and endochondral ossification, owing to the maturational arrest of osteoblasts, indicating that Cbfa1 has a fundamental role in osteoblast differentiation. However, Cbfa1 was also expressed in chondrocytes, and its expression was increased according to the maturation of chondrocytes. Terminal hypertrophic chondrocytes expressed Cbfa1 extensively. The significant expression of Cbfa1 in hypertrophic chondrocytes was first detected at embryonic day 13.5 (E13.5), and its expression in hypertrophic chondrocytes was most prominent at E14.5–16.5. In Cbfa1‐deficient mice, whose entire skeleton was composed of cartilage, the chondrocyte differentiation was disturbed. Calcification of cartilage occurred in the restricted parts of skeletons, including tibia, fibula, radius, and ulna. Type X collagen, BMP6, and Indian hedgehog were expressed in their hypertrophic chondrocytes. However, osteopontin, bone sialoprotein, and collagenase 3 were not expressed at all, indicating that they are directly regulated by Cbfa1 in the terminal hypertrophic chondrocytes. Chondrocyte differentiation was severely disturbed in the rest of the skeleton. The expression of PTH/PTHrP receptor, Indian hedgehog, type X collagen, and BMP6 was not detected in humerus and femur, indicating that chondrocyte differentiation was blocked before prehypertrophic chondrocytes. These findings demonstrate that Cbfa1 is an important factor for chondrocyte differentiation. Dev Dyn 1999;214:279–290.

Diagnostic criteria for primary osteoporosis: year 2012 revision

In 1995, the Japanese Society for Bone and Mineral Metabolism (now the Japanese Society for Bone and Mineral Research) established the Osteoporosis Diagnostic Criteria Review Committee. Following discussion held at the 13th scientific meeting of the Society in 1996, the Committee, with the consensus of its members, proposed diagnostic criteria for primary osteoporosis. The Committee revised those criteria in 1998 and again in 2000. The Japanese Society for Bone and Mineral Research and Japan Osteoporosis Society Joint Review Committee for the Revision of the Diagnostic Criteria for Primary Osteoporosis aimed at obtaining international consistency and made a revised edition based on the new findings in 2012.

Disruption of a long-range cis-acting regulator for Shh causes preaxial polydactyly

Preaxial polydactyly (PPD) is a common limb malformation in human. A number of polydactylous mouse mutants indicate that misexpression of Shh is a common requirement for generating extra digits. Here we identify a translocation breakpoint in a PPD patient and a transgenic insertion site in the polydactylous mouse mutant sasquatch (Ssq). The genetic lesions in both lie within the same respective intron of the LMBR1/Lmbr1 gene, which resides ≈1 Mb away from Shh. Genetic analysis of Ssq reveals that the Lmbr1 gene is incidental to the phenotype and that the mutation directly interrupts a cis-acting regulator of Shh. This regulator is most likely the target for generating PPD mutations in human.

Number of osteoprogenitor cells in human bone marrow markedly decreases after skeletal maturation.

Abstract: Pluripotent mesenchymal stem cells in bone marrow differentiate to osteoblast progenitor cells. When the bone marrow cells are cultured in vitro, they form colony-forming units-fibroblastic (CFU-Fs) with exhibiting osteoblastic features such as expression of alkaline phosphatase (ALP) and formation of calcified nodules ex vivo. This article describes the effect of growth, maturation, and aging of the skeleton on human CFU-Fs harvested from human iliac bone marrow. Human bone marrow cells were harvested from the ilia of 49 women, and were cultured ex vivo for examination. The 49 subjects ranged in age from 4 to 88 years and were without metabolic bone disease. These aspirated bone marrow cells from human ilium exhibited osteoblastic phenotype such as alkaline phosphatase (ALP) activity, expression of osteocalcin (OSC) and parathyroid hormone-receptor (PTH-R) mRNA, and the formation of calcified nodules in vitro. The number of ALP-positive CFU-Fs and the ALP activity were quantified. The highest levels of ALP-positive CFU-Fs were observed in the young group, particularly in those under 10 years of age. The levels of ALP-positive CFU-Fs declined sharply after 10 years of age; those above 20 years of age exhibited a lower number of ALP-positive CFU-Fs, with a gradual decline with increasing age. These results indicate that change in the number of ALP-positive CFU-Fs may be associated with skeletal growth and maturation. The results also show that osteoblastic features such as ALP activity and capability of formation of calcification nodules were maintained even in the older subjects. These findings suggest that decreased activity of bone formation in the aged subjects could be, in part, caused by the decreased number of osteoprogenitor cells differentiating into osteoblasts because the number of ALP-positive CFU-Fs was one of the indices exhibiting bone-forming activity in the human marrow stromal cells.

Common variants at SCN5A-SCN10A and HEY2 are associated with Brugada syndrome, a rare disease with high risk of sudden cardiac death

Brugada syndrome is a rare cardiac arrhythmia disorder, causally related to SCN5A mutations in around 20% of cases. Through a genome-wide association study of 312 individuals with Brugada syndrome and 1,115 controls, we detected 2 significant association signals at the SCN10A locus (rs10428132) and near the HEY2 gene (rs9388451). Independent replication confirmed both signals (meta-analyses: rs10428132, P = 1.0 × 10−68; rs9388451, P = 5.1 × 10−17) and identified one additional signal in SCN5A (at 3p21; rs11708996, P = 1.0 × 10−14). The cumulative effect of the three loci on disease susceptibility was unexpectedly large (Ptrend = 6.1 × 10−81). The association signals at SCN5A-SCN10A demonstrate that genetic polymorphisms modulating cardiac conduction can also influence susceptibility to cardiac arrhythmia. The implication of association with HEY2, supported by new evidence that Hey2 regulates cardiac electrical activity, shows that Brugada syndrome may originate from altered transcriptional programming during cardiac development. Altogether, our findings indicate that common genetic variation can have a strong impact on the predisposition to rare diseases.

Increased bone formation by intermittent parathyroid hormone administration is due to the stimulation of proliferation and differentiation of osteoprogenitor cells in bone marrow

In order to examine the mechanism of the anabolic effect of parathyroid hormone (PTH) on bone formation, human PTH(1-34) [hPTH(1-34)] (30 micrograms/kg) was injected subcutaneously to 9-week-old rats 5 times a week for 1 or 3 weeks. Trabecular bone volume (BV/TV) in the tibial metaphysis was not significantly different between the PTH- and vehicle-treated groups, but the parameters related to bone formation, including osteoid surface (OS/BS), mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS), were significantly increased as early as 1 week after PTH treatment. And the parameters related to bone resorption including eroded surface (ES/BS) and osteoclast number (N.Oc/BS) were also significantly increased as early as 1 week after PTH treatment. Treatment with PTH for 1 week induced no significant increase in bone mineral density at the femoral metaphysis, whereas the same treatment for 3 weeks induced a significant increase. When bone marrow cells isolated from femora and tibiae of either PTH- or vehicle-treated rats were cultured at a high density (2 x 10(7) cells/one well of 24-multiwell plate), cellular alkaline phosphatase (ALP) activity was significantly increased in the cells isolated from PTH-treated rats compared with vehicle-treated rats. When bone marrow cells were cultured at a low density (4 x 10(6) cells/a one well of 6-multiwell plate) to generate colonies (colony forming unit-fibroblastic, CFU-F), PTH induced apparent increases in both the total number of CFU-F and the number of ALP-positive CFU-F.(ABSTRACT TRUNCATED AT 250 WORDS)

Homeobox protein Msx2 acts as a molecular defense mechanism for preventing ossification in ligament fibroblasts

ABSTRACT Ligaments and tendons are comprised of tough yet flexible connective tissue. Little is known, however, about the precise characteristics of the cells in ligaments and tendons due to the absence of specific markers and cell lines. We recently reported a periodontal ligament cell line, PDL-L2, with suppressed Runx2/Osf2 transcriptional activity and an inability to form mineralized nodules. The present study demonstrates that the homeobox protein Msx2 is a key factor in suppressing those two functions. Msx2 colocalizes with Runx2/Osf2 and suppresses its activity cooperatively, acting with another corepressor, TLE1, as a complex to recruit histone deacetylase 1 activity. Reverse transcription-PCR and in situ hybridization demonstrated that Msx2 expression is higher in periodontal ligament and tendon cells than in osteoblasts. Stable reduction of Msx2 expression in PDL-L2 cells induces osteoblastic differentiation, thereby causing matrix mineralization. Conversely, stable, forced Msx2 expression in MC3T3-E1 cells prevented osteoblast differentiation and matrix mineralization. Msx2-induced suppression of osteoblast differentiation was repressed by bone morphogenetic protein 2. In addition, Msx2 was downregulated in a symptom- and calcification-dependent manner at the affected region in patients with ossification of the posterior longitudinal ligament. Our findings indicate that Msx2 plays a central role in preventing ligaments and tendons from mineralizing.

A Morphometric Comparison of Trabecular Structure of Human Ilium Between Microcomputed Tomography and Conventional Histomorphometry

Abstract. Recently, an imaging technique using microcomputed tomography (micro-CT) has emerged as a method for nondestructively assessing the microarchitecture of unprocessed surgical bone biopsy specimens. Using micro-CT, two-dimensional (2D) axial images were obtained from undecalcified transiliac bone biopsies which were taken from 15 patients with various metabolic bone diseases. Total area, bone area, and bone perimeter were determined, from which the bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were calculated semiautomatically and instantaneously. To evaluate the validity of this technique as a useful tool, the results were compared with those obtained from conventional histomorphometry. There were significant correlations between the two techniques for all parameters, with correlation coefficients ranging from 0.759 (Tb.N, P < 0.005) to 0.949 (BV/TV, P < 0.0001). Different resolutions seem to lead to major differences in perimeter values measured by the two methods. These factors may explain why the correlation coefficients of Tb.N and Tb.Th estimated from the perimeter and area is lower than that of BV/TV. Our results show that the micro-CT based on 2D images is a useful tool for imaging and nondestructively quantifying the microarchitecture of trabecular bone in unprocessed surgical bone specimens.

Expression of metalloproteinase-13 (Collagenase-3) is induced during fracture healing in mice.

In fracture healing, a large amount of cartilage is formed, then rapidly replaced by osseous tissue. This process requires the transition of extracellular matrix component from type II to type I collagen. We investigated the expression of matrix metalloproteinase-13 (MMP-13), which has a high potential to cleave type II as well as type I collagen, during fracture repair in mouse ribs. In situ hybridization demonstrated that MMP-13 mRNA was present throughout the healing process. It was detected in the cells of the periosteum at day 1. As fracture callus grew, strong MMP-13 mRNA signals were detected in cells of the cartilaginous callus. In the reparative and remodeling phases, both hypertrophic chondrocytes and immature osteoblastic cells in the fracture callus expressed MMP-13 mRNA strongly. These cells were located adjacent to tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts at the sites of cartilage/bone transition. In osteoclasts, MMP-13 expression was not detected. The level of MMP-13 mRNA peaked at day 14 postfracture by northern blotting. Immunohistochemical staining showed that MMP-13 was detected primarily in hypertrophic chondrocytes. These results indicate that MMP-13 is induced during fracture healing. The site- and cell-specific expression of MMP-13 and its enzymatic property suggest that MMP-13 initiates the degradation of cartilage matrix, resulting in resorption and remodeling of the callus. In conclusion, MMP-13 plays an important role in the healing process of fractured bone in mice.

Three-dimensional microstructural analysis of human trabecular bone in relation to its mechanical properties

The purpose of this preliminary study is to explore the relationship between elastic modulus, bone mineral density (BMD), and trabecular microstructure in three dimensions. Twenty cubes of trabecular bone were processed from two lumbar vertebrae obtained from one individual. The BMD of each cube was measured by dual-energy X-ray absorptiometry and peripheral quantitative computed tomography. Each cube was serially scanned by microcomputed tomography to produce three-dimensional data sets. By analyzing these data sets, three-dimensional trabecular microstructural indices of connectivity density and fractal dimension were calculated as well as histomorphometric parameters. The cubes were tested mechanically in a nondestructive manner for measurement of their elastic modulus. This preliminary study showed that: (1) bone mass index is correlated with mechanical properties, with coefficients of correlation ranging from 0.552 to 0.601; and (2) when controlling for BMD, no association could be detected between measures of structural complexity (connectivity density and fractal dimension) and elastic modulus in the craniocaudal direction of human vertebral bodies.