Kazuharu Irie

Matrix Mineralization as a Trigger for Osteocyte Maturation

The morphology of the osteocyte changes during the cells lifetime. Shortly after becoming buried in the matrix, an osteocyte is plump with a rich rough endoplasmic reticulum and a well-developed Golgi complex. This “immature” osteocyte reduces its number of organelles to become a “mature” osteocyte when it comes to reside deeper in the bone matrix. We hypothesized that mineralization of the surrounding matrix is the trigger for osteocyte maturation. To verify this, we prevented mineralization of newly formed matrix by administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and then examined the morphological changes in the osteocytes in rats. In the HEBP group, matrix mineralization was disturbed, but matrix formation was not affected. The osteocytes found in the unmineralized matrix were immature. Mature osteocytes were seen in the corresponding mineralized matrix in the control group. The immature osteocytes in the unmineralized matrix failed to show immunoreactivity with anti-sclerostin antibody, whereas mature osteocytes in the mineralized matrix showed immunoreactivity in both control and HEBP groups. These findings suggest that mineralization of the matrix surrounding the osteocyte is the trigger for cytodifferentiation from a plump immature form to a mature osteocyte. The osteocyte appears to start secreting sclerostin only after it matures in the mineralized bone matrix.

Macrophage migration inhibitory factor‐deficient mice are resistant to ovariectomy‐induced bone loss

A link between macrophage migration inhibitory factor (MIF) and estrogen has recently emerged. We examined the involvement of MIF in osteoporotic changes in bone after ovariectomy (OVX), and revealed that MIF‐deficient mice (MIF‐KO) were completely protected from this phenomenon. The increase in osteoclast number per bone surface and serum IL‐1β levels, which were observed in wild‐type mice after OVX, did not occur in MIF KO. Our data suggest that MIF plays an important role in the pathogenesis of postmenopausal osteoporosis, and could be a novel target for the treatment of this disease.

Transgenic Mice Overexpressing Macrophage Migration Inhibitory Factor (MIF) Exhibit High-Turnover Osteoporosis†

The bone phenotype of mice overexpressing MIF was studied. These mice showed decreased trabecular bone, increased bone formation rate, and increased MMP‐3, −9, and −13 mRNA expression in the femora and tibias. This model provides evidence of the role played by MIF in bone remodeling and balance in vivo.

Osteoclast differentiation in ectopic bone formation induced by recombinant human bone morphogenetic protein 2 (rhBMP-2).

Osteoclast differentiation in the process of ectopic bone formation induced by recombinant human bone morphogenetic protein 2 (rhBMP-2) was examined to clarify the relationship between osteoclast development and rhBMP-2-induced bone formation. A combination of rhBMP-2 with a porous microsphere (PMS) and blood clot was implanted subcutaneously on the bilateral chest muscles of rats. Tartrate-resistant acid phosphatase (TRAPase) activity, cathepsin K (cath K), and calcitonin receptor (CTR), as markers of osteoclasts and their precursors, were examined using enzyme and immunohistochemical analysis up to 7 days after implantation. Mononuclear cells positive for TRAPase, cath K, and CTR first appeared on day 3 in connective tissue surrounding the PMS after implantation of rhBMP-2. Simultaneously, alkaline phosphatase activity became detectable in mesenchymal cells in the connective tissue. Electron microscopy demonstrated some mononuclear cells with abundant mitochondria and poorly developed rough endoplasmic reticulum in the proximity of mesenchymal cells. However, there was no evidence of cartilage or bone matrix formation on day 3. Osteoclasts in various stages of development, classified by the pattern of immunoreactivity for cath K, were observed by day 7. The polarized intracellular distribution of cath K was found only in osteoclasts attached to bone matrix. In conclusion, we have demonstrated for the first time the appearance of osteoclast precursors before bone matrix formation induced by rhBMP-2, suggesting that bone matrix is not a prerequisite for osteoclast precursor recruitment. Furthermore, we suggest that differentiation into polarized functional osteoclasts is accomplished when the osteoclasts attach to the bone matrix.

Immunolocalization of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in Meckel's cartilage compared with developing endochondral bones in mice

We examined the immunolocalization of receptor activator of nuclear factor‐κB ligand (RANKL) and osteoprotegerin (OPG) in areas of resorption caused by osteoclasts/chondroclasts on embryonic days 14–16 (E14–16) in Meckels cartilage, and compared the results with those in endochondral bones in mice. Intense RANKL and OPG immunoreactivity was detected in the chondrocytes in Meckels cartilage. On E15, when the incisor teeth were closest to the middle portion of Meckels cartilage, tartrate‐resistant acid phosphatase (TRAP)‐positive cells appeared on the lateral side of the cartilage. Furthermore, the dental follicle showed moderate immunoreactivity for RANKL and OPG, whereas osteoblasts derived from perichondral cells were immunonegative for RANKL and OPG in that area. On E16, cartilage resorption by TRAP‐positive cells had progressed at the differential position, and intensely immunoreactive products of RANKL were overlapped on and found to exist next to TRAP‐positive cells in the resorption area. In developing metatarsal tissue, OPG immunoreactivity was intense in periosteal osteoblasts, whereas RANKL was only faintly seen in some of the periosteal cells. In epiphyseal chondrocytes of the developing femur, RANKL immunoreactivity was moderate, and OPG scarcely detected. These results indicate a peculiarity of RANKL and OPG immunolocalization in resorption of Meckels cartilage. Growth of the incisor teeth may be involved in the time‐ and position‐specific resorption of Meckels cartilage through local regulation of the RANKL/OPG system in dental follicular cells and periosteal osteoblasts, whereas RANKL and OPG in chondrocytes seem to contribute to resorption through regulation of the chondroclast function.

Role of stromal cells in osteoclast differentiation in bone marrow

Abstract. Bone marrow stromal cells have been considered to play an important role in osteoclast differentiation. However, the interaction of these cells in vivo has not been clearly demonstrated. To clarify this, we examined the distribution of alkaline phosphatase (ALPase) and tartrate-resistant acid phosphatase (TRAPase) activities as markers of osteoblastic and osteoclastic cells, respectively. Rat tibiae were fixed and embedded in Technovit 8100 or paraffin. ALPase and TRAPase activities were detected simultaneously on a plastic section by the azo-dye method. ALPase activity was detected on the plasma membranes of osteoblasts and some bone marrow fibroblastic stromal cells. These ALPase-positive cells were connected to each other by cytoplasmic processes, forming a cellular network in bone marrow. The ALPase activity of fibroblastic stromal cells tended to be stronger in those cells close to the bone surface than in the cells in the center of bone marrow. Reticular fibers in bone marrow were found to form a network. The ALPase-positive fibroblastic stromal cells may be reticular cells, because the localization of those cells was in accord with the localization of reticular fibers. The TRAPase-positive mononuclear cells and osteoclasts were mostly observed to be associated with the intensely ALPase-positive fibroblastic stromal cells. Immunoreactivity of osteoclast differentiation factor (ODF) was found in the fibroblastic stromal cells. These findings suggest that the network of ALPase-positive fibroblastic stromal cells in bone marrow serves as a guide for the migration of osteoclast precursor cells toward the bone surface, and may control the differentiation and activity of osteoclasts.

Effects of running exercise on the mandible and tibia of ovariectomized rats

Abstract To examine the effects of running exercise on the mandible and tibia of ovariectomized (OVX) rats, 26-week-old sham-operated (Sham) and OVX rats 1 week post-ovariectomy were subjected to non-exercise (Sham-Cont and OVX-Cont) and exercise (Sham-Exc and OVX-Exc) for 8 weeks. OVX induced a significant decrease in alkaline phosphatase (ALP) and an increase in tartrate-resistant acid phosphatase (TRAP) activity and a reduction of 17β-estradiol in the serum. In OVX-Cont rats, histology and bone mineral density (BMD) showed bone loss in the proximal tibia, and histology, soft X-ray photographs and bone marrow area (BMA) revealed enlargement of the bone marrow cavity in the neck of the condylar process. In OVX-Exc rats, exercise significantly increased ALP activity, decreased TRAP activity and markedly elevated serum progesterone levels. Histology and BMD in the tibia and histology, X-ray photographs and BMA in the mandible were comparable to those in Sham rats. In Sham-Exc rats, unexpected decreases were observed in serum enzymes and hormones, but the histology and BMD in the tibia and histology, X-ray photographs and BMA in the mandible were very similar to those in Sham-Cont rats, suggesting a decrease of bone turnover with no change of bone mass in the tibia and mandible. We conclude that exercise has a beneficial effect not only on bone loss in the tibia, but also on differential changes in the neck of the condylar process, perhaps by increasing serum levels of progesterone in OVX rats.

Gene Expression and Accumulation of Fibrillin-1, Fibrillin-2, and Tropoelastin in Cultured Periodontal Fibroblasts

The elastic system fibers consist of three types—oxytalan, elaunin, and elastic fibers—differing in their relative microfibril and elastin contents. All three types are found in human gingiva, but human periodontal ligaments contain only elastin-free fibers. We examined cultured human gingival fibroblasts (HGF) and cultured human periodontal ligament fibroblasts (HPLF) to determine the gene expression of fibrillin-1 and fibrillin-2 (the major components of microfibrils) and of tropoelastin. In addition, we assessed the degree of accumulation of these proteins in the extracellular matrix. Northern blot analysis revealed that the level of expression of fibrillin-1 and fibrillin-2 was higher in HGF than in HPLF. However, examination of matrix samples from HGF and HPLF cell layers showed that there was no difference in fibrillin-1 accumulation, although fibrillin-2 accumulated to a much greater extent in the HGF-derived matrix. Tropoelastin was expressed only in and around HGF. These results show a correlation between gene expression and the accumulation of tropoelastin and fibrillin-2 in HGF.

Induction of fibulin-5 gene is regulated by tropoelastin gene, and correlated with tropoelastin accumulation in vitro

Fibulin-5 (also known as DANCE) is an elastin-binding protein that is thought to play a role in elastogenesis. We examined the relationship between the gene expression of fibulin-5 and the gene expression and accumulation of tropoelastin by comparing elastin-producing cells (human gingival fibroblasts) with non-elastin-producing cells (human periodontal ligament fibroblasts) by Northern blot analysis. Fibulin-5 gene induction was found only in elastin-producing cells. Induction of the fibulin-5 gene in elastin-producing cells occurred after induction of the tropoelastin gene, and the fibulin-5 level was reduced upon RNA interference-mediated down-regulation of tropoelastin. Fibulin-5 gene induction was also correlated with a rapid increase of tropoelastin accumulation within the cell layer. These results may suggest that the fibulin-5 gene induction is directly or indirectly regulated by tropoelastin gene expression and plays a role in the accumulation of elastic fibers within matrices.

Contributions of matrix metalloproteinases toward Meckel’s cartilage resorption in mice: immunohistochemical studies, including comparisons with developing endochondral bones

The middle portion of Meckel’s cartilage (one of four portions that disappear with unique fate) degrades via hypertrophy and the cell death of chondrocytes and via the resorption of cartilage by chondroclasts. We have examined the immunolocalization of matrix metalloproteinase-2 (MMP-2), MMP-9, MMP-13, and MMP-14 (members of the MMP activation cascade) and galectin-3 (an endogenous substrate for MMP-9 and an anti-apoptotic factor) during resorption of Meckel’s cartilage in embryonic mice and have compared the results with those of developing endochondral bones in hind limbs. MMP immunoreactivity, except for MMP-2, is present in nearly all chondrocytes in the middle portion of Meckel’s cartilage. On embryonic day 15 (E15), faint MMP-2-immunoreactive and intense MMP-13-immunoreactive signals occur in the periosteal bone matrix deposited by periosteal osteoblasts on the lateral surface, whereas MMP-9 and MMP-14 are immunolocalized in the peripheral chondrocytes of Meckel’s cartilage. The activation cascade of MMPs by face-to-face cross-talk between cells may thus contribute to the initiation of Meckel’s cartilage degradation. On E16, immunopositive signaling for MMP-13 is detectable in the ruffled border of chondroclasts at the resorption front, whereas immunostaining for galectin-3 is present at all stages of chondrocyte differentiation, especially in hypertrophic chondrocytes adjacent to chondroclasts. Galectin-3-positive hypertrophic chondrocytes may therefore coordinate the resorption of calcified cartilage through cell-to-cell contact with chondroclasts. In metatarsal specimens from E16, MMPs are detected in osteoblasts, young osteocytes, and the bone matrix of the periosteal envelope, whereas galectin-3 immunoreactivity is intense in young periosteal osteocytes. In addition, intense MMP-9 and MMP-14 immunostaining has been preferentially found in pre-hypertrophic chondrocytes, although galectin-3 immunoreactivity markedly decreases in hypertrophic chondrocytes. These results indicate that the degradation of Meckel’s cartilage involves an activation cascade of MMPs that differs from that in endochondral bone formation.