Tadaharu Kawawa

Interleukin‐4 inhibition of osteoclast differentiation is stronger than that of interleukin‐13 and they are equivalent for induction of osteoprotegerin production from osteoblasts

Interleukin (IL)‐4 and IL‐13 are closely related cytokines known to inhibit osteoclast formation by targeting osteoblasts to produce an inhibitor, osteoprotegerin (OPG), as well as by directly targeting osteoclast precursors. However, whether their inhibitory actions are the same remains unclear. The inhibitory effect of IL‐4 was stronger than that of IL‐13 in an osteoclast‐differentiation culture system containing mouse osteoblasts and osteoclast precursors. Both cytokines induced OPG production by osteoblasts in similar time‐ and dose‐dependent manners. However, IL‐4 was stronger in direct inhibition that targeted osteoclast precursors. Furthermore, IL‐4 induced phosphorylation of signal transducer and activator of transcription‐6 (STAT6) at lower concentrations than those of IL‐13 in osteoclast precursors. IL‐4 but not IL‐13 strongly inhibited the expression of nuclear factor of activated T‐cells, cytoplasmic 1 (nuclear factor‐ATc1), a key factor of osteoclast differentiation, by those precursors. Thus, the activities of IL‐4 and IL‐13 toward osteoclast precursors were shown to be different in regards to inhibition of osteoclast differentiation, whereas those toward osteoblasts for inducing OPG expression were equivalent.

Identification and Characterization of the Precursors Committed to Osteoclasts Induced by TNF-Related Activation-Induced Cytokine/Receptor Activator of NF-κB Ligand

Osteoclasts are terminally differentiated from cells of monocyte/macrophage lineage by stimulation with TNF-related activation-induced cytokine (TRANCE) (receptor activator of NF-κB ligand/osteoprotegerin ligand/osteoclast differentiation factor/TNFSF11/CD254). In the present study, we attempted to determine when and how the cell fate of precursors becomes committed to osteoclasts following TRANCE stimulation. Although mouse bone marrow-derived macrophages (BMMs) were able to differentiate into either osteoclasts or dendritic cells, the cells no longer differentiated into dendritic cells after treatment with TRANCE for 24 h, indicating that their cell fate was committed to osteoclasts. Committed cells as well as BMMs were still quite weak in tartrate-resistant acid phosphatase activity, an osteoclast marker, and incorporated zymosan particles by phagocytosis. Interestingly, committed cells, but not BMMs, could still differentiate into osteoclasts even after incorporation of the zymosan particles. Furthermore, IL-4 and IFN-γ, potent inhibitors of osteoclast differentiation, failed to inhibit osteoclast differentiation from committed cells, and blocking of TRANCE stimulation by osteoprotegerin resulted in cell death. Adhesion to culture plates was believed to be essential for osteoclast differentiation; however, committed cells, but not BMMs, differentiated into multinucleated osteoclasts without adhesion to culture plates. Although LPS activated the NF-κB-mediated pathway in BMMs as well as in committed cells, the mRNA expression level of TNF-α in the committed cells was significantly lower than that in BMMs. These results suggest that characteristics of the committed cells induced by TRANCE are distinctively different from that of BMMs and osteoclasts.

Bone wound healing after maxillary molar extraction in ovariectomized aged rats: Quantitative backscattered electron image analysis

The processes of bone wound healing after maxillary molar extraction in ovariectomized aged rats were examined by means of quantitative backscattered electron image analysis and energy‐dispersive X‐ray microanalysis. Six‐month‐old female rats were either sham‐operated or underwent bilateral ovariectomy (OVX), and 60 days postoperatively, the maxillary first molars were extracted. On post‐extraction days 7, 30, and 60, the dissected and resin‐embedded maxillae were micromilled in the transverse direction through the extracted alveolar sockets, and new bone formation on the buccal maxillary bone surface and within the extracted alveolar sockets was examined. In both sham‐operated control and OVX rats, new bone formation was recognized on the buccal bone surface, as well as within the extracted sockets, and increased daily through to day 60. In comparison to sham‐operated controls, new bone formation in OVX rats was significantly decreased both on the buccal bone surface and within the extracted sockets. Our results suggest that bone wound healing by new bone formation after maxillary molar extraction is significantly decreased in OVX‐induced osteoporosis. Anat Rec 259:76–85, 2000.

Stimulation of the locus coeruleus suppresses trigeminal sensorimotor function in the rat

The nucleus locus coeruleus (LC) has been implicated in the modulation of the spinal sensorimotor function. The aim of the present study was to examine the effect of electrical stimulation of the LC on sensorimotor function in the trigeminal system. The following two cases of sensorimotor behaviors mediated by the trigeminal brainstem sensory nuclear complex were examined: (1) the activity of the masseter muscle evoked by pressure on the region of the temporomandibular joint (TMJ); and (2) the activity of the digastric muscle evoked by electrical stimulation of the tooth pulp, resulting in the jaw-opening reflex. In the first case, LC stimulation at 10, 30 and 50 microA resulted in a 70%, 68% and 55% reduction in the magnitude of electromyogram (EMG) activity of the masseter muscle compared with the control (without LC stimulation), respectively. The threshold intensity for the onset of masseter EMG activity increaced to 106%, 111% and 121% of the control with 10, 30 and 50 microA LC stimulation, respectively. In the second case, EMG magnitude in response to the digastric muscle decreased to 42% of the control when 30 microA of LC stimulation was delivered. These results suggest that descending influences from the LC can act in suppression of the trigeminal sensorimotor function.

Involvement of reticular neurons located dorsal to the facial nucleus in activation of the jaw-closing muscle in rats

The location of excitatory premotor neurons for jaw-closing motoneurons was examined by the use of electrical and chemical stimulation and extracellular single-unit recording techniques in the anesthetized rat. Single-pulse electrical stimulation of the supratrigeminal region (SupV) and the reticular formation dorsal to the facial nucleus (RdVII) elicited masseter EMG response at mean (+/-SD) latencies of 2.22 +/- 0.59 ms and 3.10 +/- 1.14 ms, respectively. Microinjection (0.1-0.3 microl) of glutamate (50 mM) or kainate (0.5-100 microM) into RdVII increased masseter nerve activity in artificially ventilated and immobilized rats by 30.2 +/- 40.5% and 50.7 +/- 46.8% compared to baseline values, respectively. Forty reticular neurons were antidromically activated by stimulation of the ipsilateral trigeminal motor nucleus (MoV). Twenty neurons were found in RdVII, and the remaining 20 neurons were located in SupV, or areas adjacent to SupV or RdVII. Eleven neurons in RdVII responded to at least either passive jaw opening or light pressure applied to the teeth or tongue. Nine neurons responded to passive jaw opening. Five of the nine neurons responded to multiple stimulus categories. A monosynaptic excitatory projection from one neuron in RdVII was detected by spike-triggered averaging of the rectified masseter nerve activity. We suggest that reticular neurons in RdVII are involved in increasing masseter muscle activity and that excitatory premotor neurons for masseter motoneurons are likely located in this area. RdVII could be an important candidate for controlling activity of jaw-closing muscles via peripheral inputs.

Thalamic- and cerebellar-projecting interpolaris neuron responses to afferent inputs

Thalamic- and cerebellar-projecting interpolaris neuron responses to afferent inputs from the temporomandibular joint (TMJ) and/or the masseter muscle (Mm) were examined in rats. Of 230 neurons tested, 24 could be antidromically stimulated from the contralateral ventral posteromedial thalamic nucleus (VPM), and 27 of 91 neurons tested were stimulated from the ipsilateral posteromedial part of crus II of the cerebellar cortex. None had dual projections. The thalamic-projecting neurons were recorded in the dorsomedial region of the interpolaris; most cerebellar-projecting neurons were at the medial border of the interpolaris. Ten of 24 thalamic- and 17 of 27 cerebellar-projecting neurons received nociceptive information. Afferent inputs from the TMJ and the Mm converged on 6 of 24 thalamic-projecting neurons and on 16 of 27 cerebellar-projecting neurons. In both the thalamic- and cerebellar-projecting neurons, there was no difference between the non-nociceptive and nociceptive neurons in mean antidromic latency. The results suggest that the interpolaris integrates and relays afferent inputs from deep oral structures.

Accuracy of titanium cast crowns obtained from calcia base mold

A calcia base investment has high stability even with melted titanium and, therefore, can produce an excellent titanium casting. In this study, titanium powder was distributed to a calcia base investment as an expanding agent, and the firing temperature of the mold was controlled at 800 degrees C. The calcia base investment with 6.1% wt titanium powder expanded 1.7% during 2 h heating at 800 degrees C. The marginal discrepancies between the die and the titanium crown were improved by the addition of the titanium powder to the investment. The mean thickness of the cement layer between the epoxy teeth and the crown using 6.1% wt titanium powder content was from 40-80 microns.

The role of macrophages in the disappearance of Meckel’s cartilage during mandibular development in mice

Meckels cartilage is a supporting tissue in the embryonic mandible that disappears during development; however, the precise mechanisms of this disappearance process are still undetermined. In this study, we observed morphological changes of Meckels cartilage with development and analyzed the factors which might be related to this process. Meckels cartilage of ICR strain mice from 14 to 19 days gestation (E14-19) were used in this study. Histological and immunohistochemical studies indicated the decrease in the amount of sulfated glycoconjugates and the localization of type I collagen in the Meckels cartilage matrix during development. Chondrocytes also expressed high acid phosphatase activities at these stages. An organ culture study indicated that Meckels cartilage at E17 disappeared during the cultivation period, while the cartilage at E14 did not disappear. Massive penetration of macrophages into the perichondrium was detected at E16. RT-PCR analysis of Meckels cartilage indicated the expression of interleukin-1β, type I collagen, MMP-9 at E17, but not at E14. MIP-1α, the candidate molecule for macrophage chemoattractant factor, was expressed at E14. These results indicated the dynamic matrix changes of Meckels cartilage during development and suggested that the functional changes of chondrocytes in synthesis of type I collagen might be induced by interleukin-1β secreted by the penetrating macrophages.

Coeruleotrigeminal suppression of nociceptive sensorimotor function during inflammation in the craniofacial region of the rat

Descending action from the locus coeruleus (LC) on the trigeminal sensorimotor function was evaluated in a rat model of oral-facial inflammation. For the induction of oral-facial inflammation, mustard oil (20% solution in 20microl mineral oil) was injected into the region of the temporomandibular joint (TMJ). One week before testing, rats received bilateral lesions of the LC using a cathodal current. The electromyogram (EMG) threshold, which is the threshold intensity for the onset of EMG activity of the masseter muscle evoked by pressure on the TMJ region, was used in the present study as an indicator of the trigeminal sensorimotor function. Following mustard oil injection, in the LC-lesioned rats, EMG thresholds significantly decreased at 30min, which lasted up to 240min. In contrast, EMG thresholds in the LC-intact rats returned to the level before injection after 180min. Systemic naloxone (1.3mg/kg, i.v.) produced a further decrease of EMG thresholds in both the LC-intact and LC-lesioned rats. Under the existence of naloxone, EMG thresholds in the LC-lesioned rats were significantly lower than those of the LC-intact rats. These results suggest that oral-facial inflammation activates the coeruleotrigeminal modulating system and that an action of this system is independent of the opioid depressive mechanism.

Influence of Deacetylated Chitin 50-TYPE I Collagen Complex Gel to Bone Marrow-Derived Mesenchymal Cells

目的: 骨組織再生のための新たな生体材料の開発や成長因子の応用が期待されている. そこで, 本研究は各種成長因子の担体として高分子複合化材料の作製を行い, この材料の骨髄由来未分化間葉系細胞への影響を検討した.方法: 脱アセチル化キチン50とType I-Aコラーゲンをクーロン反応させることで, DAC50-TYPEIコラーゲン複合化ゲルを作製した. そして, この材料内にて骨髄由来未分化間葉系細胞の三次元培養を試み, 細胞増殖そして分化という観点から細胞増殖性試験, H-E染色による形態学的観察, ALP染色によるALP活性の観察およびRT-PCR法によるBSP, ALP, Osteopontin, Osteocalcin, およびGAPDH遺伝子の発現を検索した.結果: 細胞増殖性試験では細胞増殖は認めたが, 著しい増殖傾向を示すものはなかった. H-E染色では三次元的な細胞伸展所見を呈した. ALP染色では明らかなALP陽性所見が観察された. また, RTPCR法ではOsteocalcin以外の標的遺伝子の発現を確認した.結論: 今回作製した材料は骨髄由来未分化間葉系細胞に対して為害作用がなく, 三次元培養を可能とし, 骨組織再生への新たな生体材料として期待できる物質であることが示唆された.