Tatsuya Shibata

A TNF receptor loop peptide mimic blocks RANK ligand–induced signaling, bone resorption, and bone loss

Activating receptor activator of NF-kappaB (RANK) and TNF receptor (TNFR) promote osteoclast differentiation. A critical ligand contact site on the TNFR is partly conserved in RANK. Surface plasmon resonance studies showed that a peptide (WP9QY) that mimics this TNFR contact site and inhibits TNF-alpha-induced activity bound to RANK ligand (RANKL). Changing a single residue predicted to play an important role in the interaction reduced the binding significantly. WP9QY, but not the altered control peptide, inhibited the RANKL-induced activation of RANK-dependent signaling in RAW 264.7 cells but had no effect on M-CSF-induced activation of some of the same signaling events. WP9QY but not the control peptide also prevented RANKL-induced bone resorption and osteoclastogenesis, even when TNFRs were absent or blocked. In vivo, where both RANKL and TNF-alpha promote osteoclastogenesis, osteoclast activity, and bone loss, WP9QY prevented the increased osteoclastogenesis and bone loss induced in mice by ovariectomy or low dietary calcium, in the latter case in both wild-type and TNFR double-knockout mice. These results suggest that a peptide that mimics a TNFR ligand contact site blocks bone resorption by interfering with recruitment and activation of osteoclasts by both RANKL and TNF.

S100A4: A novel negative regulator of mineralization and osteoblast differentiation

S100A4 is an intracellular calcium‐binding protein expressed by osteoblastic cells. However, its roles in bone physiology are unknown. Because before matrix mineralization, its expression is markedly diminished, we hypothesized that S100A4 negatively regulates the mineralization process. In this study, we investigated the effects of the inhibition of S100A4 synthesis on osteoblast differentiation and in vitro mineralized nodule formation. Inhibition of S100A4 synthesis was achieved by an antisense approach in the mouse osteoblastic cell line MC3T3‐E1. Cell clones that synthesized low levels of S100A4 (AS clones) produced markedly increased number of mineralized nodules at much earlier stages in comparison with controls as demonstrated by Alizarin red S and von Kossa staining. The expression of type I collagen (COLI) and osteopontin (OPN) increased in AS clones compared with controls. Bone sialoprotein (BSP) and osteocalcin (OCN), molecules associated with mineralization and markers for mature osteoblastic phenotype, were expressed in AS clones before their detection in controls. Because S100A4 was not localized in the nucleus of MC3T3‐E1 cells and AS clones, it is unlikely that S100A4 directly regulates the expression of these genes. Moreover, the expression of Cbfa1/Osf‐2 and Osx, transcription factors necessary for the expression of osteoblast‐associated genes, remained unchanged in AS clones, indicating that S100A4 may be downstream to these transcription factors. These findings indicate that S100A4 is a novel negative regulator of matrix mineralization likely by modulating the process of osteoblast differentiation.

Long-term effects of local pretreatment with alendronate on healing of replanted rat teeth.

BACKGROUND AND OBJECTIVE Our previous study showed that topical alendronate, an inhibitor of bone resorption, reduces root resorption and ankylosis for 21 d after replantation of rat teeth. The aim of the present study was to evaluate the long-term inhibitory effects of topical alendronate in the replanted teeth. MATERIAL AND METHODS The rat maxillary first molars were extracted, placed in saline containing 1 mm alendronate (alendronate group) or saline (saline group) for 5 min and then replanted. The maxillae were dissected at 60 and 120 d. Microcomputed tomography horizontal sections at three root levels were analyzed for root and bone resorption, ankylosis and pulp mineralization. RESULTS In the alendronate group at 60 and 120 d, the frequencies of resorption of roots and bone were lower than those in the saline group. The p values show statistical significances of lower frequencies in the alendronate group than in the saline group by chi-square test (see Table 1). Ankylosis and pulp mineralization occurred in the alendronate and saline groups. Bone marrow spaces were narrowed in conjunction with bone tissue expansion around the replanted teeth in the alendronate group. CONCLUSION The inhibitory effects of topical alendronate were retained on root and bone resorption, but not on ankylosis and pulp mineralization, in the replanted teeth for 4 mo. Alendronate might also stimulate bone formation around the rat replanted teeth.

Improved methods for immunohistochemical detection of BrdU in hard tissue

Bromodeoxyuridine (BrdU) is used to label synthesizing DNA and to chase label-retaining cell (LRC). As stem cells divide slowly in adult tissues, they can be visualized as LRCs. In order to identify LRCs in hard tissue, we examined optimal conditions of fixation, demineralization, and DNA denaturation/antigen retrieval for immunohistochemistry of BrdU in hard tissues including bone, tooth, and periodontal ligament. Mice were subcutaneously injected with BrdU (50 microg/g body weight) twice a day from the postnatal day 11 to day 15 and sacrificed at 2 h after the last injection. Dissected maxillae were fixed (Bouins solution or 4% paraformaldehyde), demineralized (Morses solution or EDTA), and embedded in paraffin. Antigen retrieval procedures were performed before incubation with primary antibody. When sections were treated with HCl for DNA denaturation, the staining intensity of BrdU positive cells was not affected by difference of fixatives. Higher sensitivity was obtained by demineralization with Morse than with EDTA. Although heat-induced antigen retrieval techniques in citrate buffer (pH 6.0) showed as well or better sensitivity than acid pretreatment, heating caused tissue damage specifically to tooth dentine and the surrounding tissue. When the LRCs at four weeks after the last injection of BrdU were compared, much more LRCs were observed in specimen demineralized with Morse than with 10% EDTA. Our data suggest that demineralization with Morse with Bouin fixative plus HCl pretreatment gives rise to the optimal results for BrdU immunodetection in hard tissue.

The effects of intrusive loading on axial movements of impeded and unimpeded rat incisors: estimation of eruptive force

Axial movements of impeded and unimpeded rat mandibular incisors were measured following application and removal of intrusive loads of 1, 2, 5, 10 and 20 mN in a stepwise order at intervals of 1h on erupting teeth. The tooth movements were recorded by a displacement detector under artificial respiration with halothane anaesthesia. The loading and unloading procedures brought about the tooth movements in two steps: an initial rapid movement immediately after application or removal of the load and a subsequent slow and gradual movement. The initial rapid intrusive or extrusive tooth movements were significantly greater in the unimpeded than in the impeded teeth at the same load. The forces to stop extrusive tooth movements, estimated from the formula of regression lines showing correlations between the intrusive loads from 0 to 5 mN and tooth movements (microm/30 min), were 4.2 mN in the impeded and 2.9 mN in the unimpeded incisors. We suggest that repeated shortenings of the rat incisor did not cause an increase in the eruptive force and that changes in the resistance of the periodontal ligament predominantly regulate the axial movement of the rat incisor.

Mechanical response of periodontal ligament: Effects of specimen geometry, preconditioning cycles and time lapse

This study was conducted as part of research line addressing the mechanical response of periodontal ligament (PDL) to tensile-compressive sinusoidal loading. The aim of the present project was to determine the effect of three potential sources of variability: (1) specimen geometry, (2) tissue preconditioning and (3) tissue structural degradation over time. For the three conditions, selected mechanical parameters were evaluated and compared. (1) Standard flat specimens (obtained by sequentially slicing portions of bone, PDL and dentin using a precision band saw) and new cylindrical specimens (extracted with a diamond-coated trephine drill) were obtained from bovine mandibular first molars and subjected to a sinusoidal load profile. (2) Specimens were loaded with up to 2000 cycles. (3) Specimens were immersed in saline and tested after 0, 30 and 60 min. From the data generated, the following was concluded: (1) specimen geometry and preparation technique do not influence the mechanical response of the PDL; (2) the mechanical response stabilizes after approximately 1000 cycles; and (3) no major structural degradation occurs when PDL is immersed in saline for a time lapse up to 60 min.

Cationized gelatin hydrogels mixed with plasmid DNA induce stronger and more sustained gene expression than atelocollagen at calvarial bone defects in vivo

Abstract Gene transduction of exogenous factors at local sites in vivo is a promising approach to promote regeneration of tissue defects owing to its simplicity and capacity for expression of a variety of genes. Gene transduction by viral vectors is highly efficient; however, there are safety concerns associated with viruses. As a method for nonviral gene transduction, plasmid DNA delivery is safer and simpler, but requires an efficient carrier substance. Here, we aimed to develop a simple, efficient method for bone regeneration by gene transduction and to identify optimal conditions for plasmid DNA delivery at bone defect sites. We focused on carrier substances and compared the efficiencies of two collagen derivatives, atelocollagen, and gelatin hydrogel, as substrates for plasmid DNA delivery in vivo. To assess the efficiencies of these substrates, we examined exogenous expression of green fluorescence protein (GFP) by fluorescence microscopy, polymerase chain reaction, and immunohistochemistry. GFP expression at the bone defect site was higher when gelatin hydrogel was used as a substrate to deliver plasmids than when atelocollagen was used. Moreover, the gelatin hydrogel was almost completely absorbed at the defect site, whereas some atelocollagen remained. When a plasmid harboring bone morphogenic protein 2 was delivered with the substrate to bony defect sites, more new bone formation was observed in the gelatin group than in the atelocollagen group. These results suggested that the gelatin hydrogel was more efficient than atelocollagen as a substrate for local gene delivery and may be a superior material for induction of bone regeneration.

Effects of an intravenous infusion of Ringer's solution on eruption rates of incisor teeth in anesthetized rats

Objective. The vasculature within the socket is reportedly involved in determining the position of continuously erupting teeth. Thus, loss of body fluid in anesthetized rats, which would affect the vascular physiology, should influence tooth movement. We investigated the effects of an infusion of Ringers solution on the systemic arterial blood pressure, regional blood flow at the base of the incisor, and axial tooth movement in anesthetized rats to determine the cause of tooth displacement. Material and methods. In the experimental group, the animals received intravenous infusions of Ringers solution at 27 µl/min for 13 h. In the control group, the animals did not receive the infusion. Results. The infusion of Ringers solution suppressed an increase of the mean arterial blood pressure from 86 to 80 mmHg and a decrease of the regional blood flow from 170 to 217 mV, and increased the eruption rate from 267 to 361 µm/13 h during the experimental period. There was a positive correlation between the eruption rate and regional blood flow, and a negative correlation between the blood pressure and regional blood flow. Conclusions. These results suggest that an infusion of Ringers solution can cause an increase in the regional blood flow, resulting in increased fluid volume, elevated intra-socket pressure, and increased eruptive movement. It is possible that the regional vascular volume and/or pressure within the socket play an important role in determining the position of the incisor.

On the mechanical testing of the bovine periodontal ligament

In this paper a comparison between standard uniaxial flat (parallelepiped-shaped) and new cylindrical specimens is reported. The new specimen has been introduced to allow for multiaxial testing of the bovine periodontal ligament (PDL). When placed in a custom-made pressure chamber, a lateral pressure can be applied to the specimen thus permitting different stress states to be realised. Such stress states are more realistic and closer to the in vivo conditions. In the present work, the cylindrical specimen is kept at atmospheric pressure so that the comparison with the standard specimen is pertinent.