Koh-ichi Kuremoto

In Situ Tissue Engineering of Periodontal Tissues by Seeding with Periodontal Ligament-Derived Cells

The feasibility of an in situ tissue-engineering method employing cell-based therapy with autologous periodontal ligament-derived cells was investigated. Periodontal ligament cells were obtained from six beagle dogs. Periodontal fenestration defects (6 x 4 mm) were created bilaterally at a location 6 mm apical to the marginal alveolar crest in the maxillary canines. Alkaline phosphatase-positive periodontal ligament cells (3 x 10(5) cells) were seeded onto a collagen sponge scaffold just before implantation. One defect was filled with the cell-scaffold construct, and another was left empty as the control. All animals were killed 4 weeks after surgery, and specimens were evaluated histomorphometrically. All the histomorphometrical data were analyzed by three-way analysis of variance with the Bonferroni multiple comparisons test. Regeneration of apical tissue was faster than that of coronal and isolated tissues on the control side (apical > coronal > isolated; p < 0.0001). On the other hand, on the cell-seeded side, regeneration of the cementum was observed uniformly on the root surface. Our data suggest that the seeded cells induced cementum regeneration on the root surface, indicating the potential of in situ tissue engineering using autologous cells for the regeneration of periodontal tissues.

Core binding factor beta functions in the maintenance of stem cells and orchestrates continuous proliferation and differentiation in mouse incisors.

Rodent incisors grow continuously throughout life, and epithelial progenitor cells are supplied from stem cells in the cervical loop. We report that epithelial Runx genes are involved in the maintenance of epithelial stem cells and their subsequent continuous differentiation and therefore growth of the incisors. Core binding factor β (Cbfb) acts as a binding partner for all Runx proteins, and targeted inactivation of this molecule abrogates the activity of all Runx complexes. Mice deficient in epithelial Cbfb produce short incisors and display marked underdevelopment of the cervical loop and suppressed epithelial Fgf9 expression and mesenchymal Fgf3 and Fgf10 expression in the cervical loop. In culture, FGF9 protein rescues these phenotypes. These findings indicate that epithelial Runx functions to maintain epithelial stem cells and that Fgf9 may be a target gene of Runx signaling. Cbfb mutants also lack enamel formation and display downregulated Shh mRNA expression in cells differentiating into ameloblasts. Furthermore, Fgf9 deficiency results in a proximal shift of the Shh expressing cell population and ectopic FGF9 protein suppresses Shh expression. These findings indicate that Shh as well as Fgf9 expression is maintained by Runx/Cbfb but that Fgf9 antagonizes Shh expression. The present results provide the first genetic evidence that Runx/Cbfb genes function in the maintenance of stem cells in developing incisors by activating Fgf signaling loops between the epithelium and mesenchyme. In addition, Runx genes also orchestrate continuous proliferation and differentiation by maintaining the expression of Fgf9 and Shh mRNA. STEM CELLS 2011;29:1792–1803

Runx1 mediates the development of the granular convoluted tubules in the submandibular glands

The mouse granular convoluted tubules (GCTs), which are only located in the submandibular gland (SMG) are known to develop and maintain their structure in an androgen-dependent manner. We previously demonstrated that the GCTs are involuted by the epithelial deletion of core binding factor β (CBFβ), a transcription factor that physically interacts with any of the Runt-related transcription factor (RUNX) proteins (RUNX1, 2 and 3). This result clearly demonstrates that the Runx /Cbfb signaling pathway is indispensable in the development of the GCTs. However, it is not clear which of the RUNX proteins plays useful role in the development of the GCTs by activating the Runx /Cbfb signaling pathway. Past studies have revealed that the Runx /Cbfb signaling pathway plays important roles in various aspects of development and homeostatic events. Moreover, the Runx genes have different temporospatial requirements depending on the biological situation. In the present study, the GCTs of the SMG showed a remarkable phenotype of, which phenocopied the epithelial deletion of Cbfb, in epithelial-specific Runx1 conditional knock-out (cKO) mice. The results indicate that Runx1 works as a partner of Cbfb during the development of the GCTs. We also discovered that the depletion of Runx1 resulted in the reduced secretion of saliva in male mice. Consistent with this finding, one of the water channels, Aquaporin-5 (AQP5) was mislocalized in the cytoplasm of the Runx1 mutants, suggesting a novel role of Runx1 in the membrane trafficking of AQP5. In summary, the present findings demonstrated that RUNX1 is essential for the development of the GCTs. Furthermore, RUNX1 could also be involved in the membrane trafficking of the AQP5 protein of the acinar cells in the SMG in order to allow for the proper secretion of saliva.

Sphingomyelin generated by sphingomyelin synthase 1 is involved in attachment and infection with Japanese encephalitis virus

Japanese encephalitis virus (JEV) is a mosquito-borne RNA virus which infects target cells via the envelope protein JEV-E. However, its cellular targets are largely unknown. To investigate the role of sphingomyelin (SM) in JEV infection, we utilized SM-deficient immortalized mouse embryonic fibroblasts (tMEF) established from SM synthase 1 (SMS1)/SMS2 double knockout mice. SMS deficiency significantly reduced both intracellular and extracellular JEV levels at 48 h after infection. Furthermore, after 15 min treatment with JEV, the early steps of JEV infection such as attachment and cell entry were also diminished in SMS-deficient tMEFs. The inhibition of JEV attachment and infection were recovered by overexpression of SMS1 but not SMS2, suggesting SMS1 contributes to SM production for JEV attachment and infection. Finally, intraperitoneal injection of JEV into SMS1-deficient mice showed an obvious decrease of JEV infection and its associated pathologies, such as meningitis, lymphocyte infiltration, and elevation of interleukin 6, compared with wild type mice. These results suggest that SMS1-generated SM on the plasma membrane is related in JEV attachment and subsequent infection, and may be a target for inhibition of JEV infection.

Runx/Cbfb signaling regulates postnatal development of granular convoluted tubule in the mouse submandibular gland

Background: The rodent salivary gland is not fully developed at birth and the cellular definitive differentiation takes place postnatally. However, little is known about its molecular mechanism. Results: Here we provide the loss‐of‐function genetic evidence that Runx signaling affects postnatal development of the submandibular gland (SMG). Core binding factor β (Cbfb) is a cotranscription factor which forms a heterodimer with Runx proteins. Cbfb was specifically expressed in the duct epithelium, specifically in the SMG. Epithelial Cbfb deficiency resulted in decrease in the size of the SMG and in the saliva secretion on postnatal day 35. The Cbfb mutant SMG specifically exhibited involution of the granular convoluted tubules (GCT), with a down‐regulated expression of its marker genes, such as Klk1, Ngf, and Egf. The induction of GCT is under the control of androgens, and the Cbfb mutant SMG demonstrated down‐regulated expression of Crisp3, an androgen‐dependent transcript. Because the circulating testosterone or tissue dihydrotestosterone levels were not affected in the Cbfb mutants, it appears that Runx/Cbfb signaling regulate androgen receptor pathway, but does not affect the circulating testosterone levels or the enzymatic conversion to DHT. Conclusions: Runx signaling is important in the postnatal development of androgen‐dependent GCT in the SMG. Developmental Dynamics 244:488–496, 2015.

Runx1-Stat3-Tgfb3 signaling network regulating the anterior palatal development

Runx1 deficiency results in an anteriorly specific cleft palate at the boundary between the primary and secondary palates and in the first rugae area of the secondary palate in mice. However, the cellular and molecular pathogenesis underlying such regional specificity remain unknown. In this study, Runx1 epithelial-specific deletion led to the failed disintegration of the contacting palatal epithelium and markedly downregulated Tgfb3 expression in the primary palate and nasal septum. In culture, TGFB3 protein rescued the clefting of the mutant. Furthermore, Stat3 phosphorylation was disturbed in the corresponding cleft regions in Runx1 mutants. The Stat3 function was manifested by palatal fusion defects in culture following Stat3 inhibitor treatment with significant downregulation of Tgfb3. Tgfb3 is therefore a critical target of Runx1 signaling, and this signaling axis could be mediated by Stat3 activation. Interestingly, the expression of Socs3, an inhibitor of Stat3, was specific in the primary palate and upregulated by Runx1 deficiency. Thus, the involvement of Socs3 in Runx1-Tgfb3 signaling might explain, at least in part, the anteriorly specific downregulation of Tgfb3 expression and Stat3 activity in Runx1 mutants. This is the first study to show that the novel Runx1-Stat3-Tgfb3 axis is essential in anterior palatogenesis.

Runx1-Stat3 signaling regulates the epithelial stem cells in continuously growing incisors

Rodent incisors grow permanently and the homeostasis of enamel production is maintained by a continuous supply of epithelial progenitors from putative stem cells in the cervical loop. We herein report that Runx1 regulates the Lgr5-expressing epithelial stem cells and their subsequent continuous differentiation into ameloblasts. Mice deficient in epithelial Runx1 demonstrate remarkable shortening of the incisors with underdevelopment of the cervical loop and enamel defects. In this mutant cervical loop, the proliferation of the dental epithelium was significantly disturbed and the expression of Lgr5 and enamel matrix proteins was remarkably downregulated. Interestingly, the expression of Socs3, an inhibitor of Stat3 signaling, was upregulated and Stat3 phosphorylation was suppressed specifically in the mutant cervical loop. The expression of Lgr5 and the enamel matrix protein in the wild-type incisor germs is disturbed by pharmaceutical Stat3 inhibition in vitro., of. Conversely, pharmaceutical activation of Stat3 rescues the defective phenotypes of the Runx1 mutant with upregulated Lgr5 and enamel matrix protein genes. The present results provide the first evidence of the role of Runx1 regulates the Lgr5-expressing epithelial stem cells and differentiation of ameloblast progenitors in the developing incisors. Our study also demonstrates that Stat3 modulates the Runx1-Lgr5 axis in the cervical loop.

Influence of Tooth Clenching on Mandibular Position and Fluctuation during Intercuspation

目的: 咬頭嵌合時の下顎は, 前後的, 左右的に約40μmの範囲で常に動いていることが明らかとなっている. 本研究は, 閉口筋の状態や咬頭嵌合状態の違いが, 咬頭嵌合時の下顎の位置と動揺にどのように関与しているかを明らかにすることを目的とした.方法: 被験者は, 個性正常咬合を有する成人有歯顎者11名とした. 実験条件は, 被験者にクレンチングを行わないよう指示し, 静かに咬頭嵌合位をとらせる条件, 被験者に咬頭嵌合位で強いクレンチングを行わせる条件, クレンチング直後に, クレンチングを行わずに, ただちに静かに咬頭嵌合位をとらせる条件の計3条件とした. これらの試行はそれぞれランダムに行い, 3回繰り返した. 得られたデータより, 下顎の水平面二次元の変動を解析し, 分散分析法にて統計学的に検討を行った.結果: クレンチング時とその直後では, 下顎は有意に前方および側方に偏位することが明らかとなった. またクレンチング時とその直後では, 下顎の動揺範囲は前後的に有意に大きくなることが明らかとなった.結論: 咬頭嵌合時の下顎の位置と動揺は, 閉口筋の収縮強さや筋疲労の状態やクレンチングによる歯の変位が大きな影響を及ぼしていることが明らかとなり, また歯の変位に伴う歯根膜感覚の異常が大きな影響を及ぼしていることが示唆された. しかしながら, 咬合接触状態や咬頭嵌合状態の緊密度, 下顎の歪みによる影響は少ないことが明らかとなった.

Fluctuation of the Mandible during Intercuspation.

目的: 咬頭嵌合時に下顎は動いていることはこれまでの報告で明らかとなっている. しかしながらその詳細についてはいまだ不明である. 本研究は咬頭嵌合時の下顎の動揺を明らかにすることを目的として, 詳細に検討を行った.方法: 本研究において, 光スポット位置検出センサと赤外線発光ダイオードを利用して計測を行った. このセンサを用いて, 被験者は個性正常咬合を有する成人有歯顎者11名を対象に, 咬合器に装着した各被験者の上下顎模型の咬頭嵌合位 (on the model) と各被験者の生体における咬頭嵌合位 (in vivo) をそれぞれ観察した. 得られたデータよりそれぞれの水平面の二次元の変動を解析し, 分散分析法にて統計学的に検討を行った.結果: 分散分析の結果, 下顎の動揺範囲において, on the modelでは前後, 左右的に平均6μmであったのに対し, in vivoでは前後, 左右的には平均39.9μmの値をとり, 測定条件間にのみ有意な差を認めた. また下顎の動揺のばらつきを示す分散においても, 前後方向, 左右方向ともにon the modelに対してin vivoは大きな値を示し, 測定条件間にのみ有意な差を認めた.結論: 正常者において, 咬頭嵌合時に下顎は前後的, 左右的に約40μm範囲で常に動揺していることが明らかとなった. またこの動揺は, 咬頭嵌合位を一定に保持しようとするための生理的な動きであると推察された.