Keiichi Ohya

Stimulation of Bone Formation in Cortical Bone of Mice Treated with A Receptor Activator of Nuclear Factor-κB Ligand (RANKL) Binding Peptide That Possesses Osteoclastogenesis Inhibitory Activity

Background: A RANKL-binding peptide WP9QY (W9) is known to inhibit osteoclastogenesis. Results: W9 showed an anabolic effect on cortical bone in mice. W9 bound RANKL and differentiated osteoblasts with production of autocrine factors like BMP-4. Conclusion: Signaling through RANKL is involved in part in the W9-induced osteoblast differentiation. Significance: The RANKL pathway could be a novel mechanism in osteoblast differentiation. To date, parathyroid hormone is the only clinically available bone anabolic drug. The major difficulty in the development of such drugs is the lack of clarification of the mechanisms regulating osteoblast differentiation and bone formation. Here, we report a peptide (W9) known to abrogate osteoclast differentiation in vivo via blocking receptor activator of nuclear factor-κB ligand (RANKL)-RANK signaling that we surprisingly found exhibits a bone anabolic effect in vivo. Subcutaneous administration of W9 three times/day for 5 days significantly augmented bone mineral density in mouse cortical bone. Histomorphometric analysis showed a decrease in osteoclastogenesis in the distal femoral metaphysis and a significant increase in bone formation in the femoral diaphysis. Our findings suggest that W9 exerts bone anabolic activity. To clarify the mechanisms involved in this activity, we investigated the effects of W9 on osteoblast differentiation/mineralization in MC3T3-E1 (E1) cells. W9 markedly increased alkaline phosphatase (a marker enzyme of osteoblasts) activity and mineralization as shown by alizarin red staining. Gene expression of several osteogenesis-related factors was increased in W9-treated E1 cells. Addition of W9 activated p38 MAPK and Smad1/5/8 in E1 cells, and W9 showed osteogenesis stimulatory activity synergistically with BMP-2 in vitro and ectopic bone formation. Knockdown of RANKL expression in E1 cells reduced the effect of W9. Furthermore, W9 showed a weak effect on RANKL-deficient osteoblasts in alkaline phosphatase assay. Taken together, our findings suggest that this peptide may be useful for the treatment of bone diseases, and W9 achieves its bone anabolic activity through RANKL on osteoblasts accompanied by production of several autocrine factors.

Peptide-based delivery to bone☆

Peptides are attractive as novel therapeutic reagents, since they are flexible in adopting and mimicking the local structural features of proteins. Versatile capabilities to perform organic synthetic manipulations are another unique feature of peptides compared to protein-based medicines, such as antibodies. On the other hand, a disadvantage of using a peptide for a therapeutic purpose is its low stability and/or high level of aggregation. During the past two decades, numerous peptides were developed for the treatment of bone diseases, and some peptides have already been used for local applications to repair bone defects in the clinic. However, very few peptides have the ability to form bone themselves. We herein summarize the effects of the therapeutic peptides on bone loss and/or local bone defects, including the results from basic studies. We also herein describe some possible methods for overcoming the obstacles associated with using therapeutic peptide candidates.

Selective inhibition of NF-κB suppresses bone invasion by oral squamous cell carcinoma in vivo†

Nuclear factor‐κB (NF‐κB) is constitutively activated in many cancers, including oral squamous cell carcinoma (OSCC), and is involved in the invasive characteristics of OSCC, such as growth, antiapoptotic activity and protease production. However, the cellular mechanism underlying NF‐κBs promotion of bone invasion by OSCC is unclear. Therefore, we investigated the role of NF‐κB in bone invasion by OSCC in vivo. Immunohistochemical staining of OSCC invading bone in 10 patients indicated that the expression and nuclear translocation of p65, a main subunit of NF‐κB, was increased in OSCC compared with normal squamous epithelial cells. An active form of p65 phosphorylated at serine 536 was present mainly in the nucleus in not only differentiated tumor cells but also tumor‐associated stromal cells and bone‐resorbing osteoclasts. We next injected mouse OSCC SCCVII cells into the masseter region of C3H/HeN mice. Mice were treated for 3 weeks with a selective NF‐κB inhibitor, NBD peptide, which disrupts the association of NF‐κB essential modulator (NEMO) with IκB kinases. NBD peptide treatment inhibited TNFα‐induced and constitutive NF‐κB activation in SCCVII cells in vitro and in vivo, respectively. Treatment with NBD peptide decreased zygoma and mandible destruction by SCCVII cells, reduced number of osteoclasts by inhibiting RANKL expression in osteoblastic cells and SCCVII cells, increased apoptosis and suppressed the proliferation of SCCVII cells. Taken together, our data clearly indicate that inhibition of NF‐κB is useful for inhibiting bone invasion by OSCC.

Enhanced osteoblast and osteoclast responses to a thin film sputtered hydroxyapatite coating

A sputtering technique followed by a low temperature hydrothermal treatment has been demonstrated to produce a dense-and-bioactive hydroxyapatite thin film coating. The purpose of the present study was to investigate osteoblast and osteoclast responses to the hydroxyapatite coated plates and titanium plates with similar roughness. Rat bone marrow stromal cells were cultured on these plates to induce osteoblasts. The cells showed a significantly enhanced proliferation on the hydroxyapatite surface, accompanied by increase of osteoblastic phenotypes. The co-cultured osteoclasts exhibited the significantly different cell number and morphology between the hydroxyapatite and the titanium surfaces. A series of osteoclast marker genes were more stimulated on the hydroxyapatite and thirty two percent of the hydroxyapatite surface area could be resorbed by osteoclasts. The thin film sputtered hydroxyapatite could provide a favorable surface for both osteoblast and osteoclast formation and their function, indicating its good osteoconductivity and biodegradability.

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 15xa0weeks 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.

Increased bone mass in adult prostacyclin-deficient mice

Prostaglandins (PGs) are key regulatory factors that affect bone metabolism. Prostaglandin E(2) (PGE(2)) regulates bone resorption and bone formation. Prostacyclin (PGI(2)) is one of the major products derived from arachidonic acid by the action of cyclooxygenase and PGI(2) synthase (PGIS). Unlike PGE(2), there are few reports about the role of PGI(2) in bone regulation. Therefore, we investigated the potential effect of PGI(2) on bone metabolism. We used PGIS knockout (PGIS(-/-)), PGIS heterozygous (PGIS(+)(/-)), and wild-type mice to investigate the role of PGI(2). Notably, PGIS(-/-) mice gradually displayed an increase in trabecular bone mass in adolescence. Adult PGIS(-/-) mice showed an increase in trabecular bone volume/tissue volume. Histomorphometric analysis showed that PGIS(-/-) mice displayed increases in both bone formation and bone resorption parameters. Levels of serum osteocalcin and C-telopeptides were increased in adult PGIS(-/-) mice. Furthermore, the increased bone mass patterns were rescued in PGIS(-)/(tg) mice. In conclusion, adult PGIS(-/-) mice displayed an overall increase in the levels of both bone formation and bone resorption parameters, which suggests that PGI(2) deficiency accelerates high bone turnover activity with a greater increase in bone mass in aging. These results indicated that PGI(2) may contribute to the maintenance of normal bone mass and micro-architecture in mice in age-dependent manner. Our findings demonstrate for the first time that PGI(2) is involved in bone metabolism in vivo.

Osteoclast and Osteoblast Activities on Carbonate Apatite Plates in Cell Cultures

Previous studies have demonstrated that carbonate apatite (CA) is superior to hydroxyapatite (HA) and β-tricalciumphosphate (β-TCP) with regard to osteoclastic resorption, but evidence on osteoclast and osteoblast response remains controversial. In the present study, the expression of bone related mRNA is examined on CA, HA, β-TCP, and titanium plates. ICR mouse osteoblast cells are cocultured with ICR mouse bone marrow cells. Crude osteoclast-like cell-rich suspensions are then seeded onto plates and cultured for 48u2009h. Total RNA is extracted and mRNA expression is examined by real-time RT-PCR. Amounts of vacuolar-type ATPase, cathepsin K, and TRAP mRNA are significantly greater on CA than on the other plates. The amount of osteoprotegerin mRNA is significantly greater on CA than on the other plates. RANKL mRNA expression, which is generally regarded as an osteoblast maker, varies with material, but shows no significant differences between CA and the other plates. The formation and activity of osteoclasts is greater with CA than with the other plates. Thus, CA is superior to β-TCP as a bioresorbable bone substitute for tissue engineering.

Defective nuclear factor‐κB‐inducing kinase in aly/aly mice prevents bone resorption induced by local injection of lipopolysaccharide

BACKGROUND AND OBJECTIVEnNuclear factor-κB (NF-κB) is activated at sites of inflammation in many diseases, including periodontitis. Nuclear factor-κB induces the transcription of proinflammatory cytokines, resulting in increased osteoclastogenesis and bone resorption. Recently, it has been shown that the NF-κB alternative pathway is important for maintainance of physiological bone homeostasis. Activation of this pathway is by processing of the inhibitor p100 into the active subunit p52 by nuclear factor-κB-inducing kinase (NIK). Defective NIK in aly/aly mice (NIK(aly)) causes mild osteopetrosis and blunted RANKL-stimulated osteoclastogenesis in vivo and in vitro, suggesting that NIK is necessary for basal and stimulated osteoclastogenesis. Nevertheless, the role of NIK in pathological bone resorption is not well investigated. The present study was undertaken to investigate the role of NIK in lipopolysaccharide (LPS)-induced inflammatory bone resorption using aly/aly mice.nnnMATERIAL AND METHODSnMice were injected with LPS over the calvariae and killed 5 d later. Calvariae were subjected to radiological analysis. Histological sections were stained for tartrate-resistant acid phosphatase, and histomorphometric analysis was performed to quantify the number of osteoclasts and the area of bone resorption.nnnRESULTSnLipopolysaccharide-induced inflammation was observed in wild-type and aly/+ mice but not in aly/aly mice. Lipopolysaccharide significantly reduced the calvarial bone mineral density in wild-type and aly/+ mice, whereas bone mineral density was comparable in LPS- and vehicle-injected aly/aly mice. In addition, aly/aly mice were resistant to LPS-induced bone resorption and osteoclastogenesis.nnnCONCLUSIONnTaken together, these data show that NIK is important in the bone-destructive components of inflammation and represents a possible therapeutic target.

The influence of mechanical stimulation on osteoclast localization in the mouse maxilla: bone histomorphometry and finite element analysis.

The mechanism of traumatic bone resorption in the denture-bearing bone has not yet been established with regard to the osteoclastic activity in relation to the mechanical stimulus. The purpose of this study was to clarify whether osteoclast appearance in maxilla depends on the strain intensity, using the murine loading model. The maxillary palate of thirteen-week-old male C57BL/6 mice was subjected to continuous pressure of 2xa0kPa (low stimulation, nxa0=xa04) or 7xa0kPa (high stimulation, nxa0=xa04) for 30xa0min/day for 7 consecutive days, and the mice were sacrificed after the last loading. The control group underwent the same protocol without load (nxa0=xa04). An animal-specific finite element model was constructed based on morphology and characteristics obtained from the micro-CT data and used to calculate the strain intensity of the bone. The bone histomorphometric technique revealed significant reduction of cortical bone volume and significant increase of bone resorption parameters such as osteoclast number in the bone tissue under the loading contact in comparison to the control (pxa0<xa00.05). The osteoclasts were observed in the subsurface region adjacent to the loading contact and the peripheral region of the marrow space in the intracortical region of the cortical bone in the mouse maxilla in both stimulation groups. An average of more than 90 % of the osteoclasts was observed in the areas with strain intensity higher than 85.0μ strain for the high stimulation group. The result suggests that the osteoclastic resorption is location-dependent and is also sensitive to the local strain intensity.

Regeneration of Bone- and Tendon/Ligament-Like Tissues Induced by Gene Transfer of Bone Morphogenetic Protein-12 in a Rat Bone Defect:

Members of the bone morphogenetic protein (BMP) family have diverse physiological roles. For instance, BMP-2 stimulates osteogenesis, while BMP-12 induces the formation of tendon/ligament-like tissues. Here, we designed a study to determine whether BMP-12 has bone and/or cartilage regeneration abilities similar to those of BMP-2. We implanted plasmid vectors encoding either BMP-2 or BMP-12 in rats with femur defects, and monitored the bone healing process for 8-weeks. The BMP-12 transgene induced prominent fibrogenesis by 2 weeks, with bone substitution occurring by 8 weeks. BMP-2, however, was associated predominantly with osteogenesis throughout the 8 week period. Thus, we conclude that BMP-12 does not function similarly to BMP-2 during bone healing. Further work is needed to better understand the mechanisms by which it stimulates bony growths to replace the connective tissues formed during the first stages of bone healing.