CHEMICALLY CROSS-LINKED HYDROGEL HAVING HIGH MECHANICAL STRENGTH

The mechanically tough shape memory gel (SMG) was synthesized. The chemically cross-linked process was applied using a cross-linker named methylenebisacrylamide (MBAA). The SMG was prepared by N, Ndimethyl acrylamide (DMAAm) and stearyl acrylate (SA). The DMAAm is a hydrophilic monomer, whereas SA is a hydrophobic monomer. Due to the transparency, shape memory property, low friction, and high water content, the SMG is suitable for biomedical and optical applications. The physical property such as mechanical strength of most hydrogels is not good enough. However, the properties of these hydrogels are easily controlled by changing the polymer concentration, molecular weight and cross-linker concentrations. It is found that the swelling degree increases with respect to the DMAAm concentration. The mechanical properties of transparent shape memory hydrogel were investigated by the tensile, the compression and the dynamic mechanical analysis. The Young’s modulus gradually decreases with the increase of hydrophilic components. On the other hand, higher Young’s modulus is observed by increasing the high hydrophobic concentration. It is also seen that the mechanical stress decreases with respect to the temperature, indicating the loss of Young’s modulus. The result of dynamic mechanical analysis designates that the water swollen sample has both the elastic and viscous properties

CHEMICALLY CROSS-LINKED HYDROGEL HAVING HIGH MECHANICAL STRENGTH

The mechanically tough shape memory gel (SMG) was synthesized. The chemically cross-linked process was applied using a cross-linker named methylenebisacrylamide (MBAA). The SMG was prepared by N, Ndimethyl acrylamide (DMAAm) and stearyl acrylate (SA). The DMAAm is a hydrophilic monomer, whereas SA is a hydrophobic monomer. Due to the transparency, shape memory property, low friction, and high water content, the SMG is suitable for biomedical and optical applications. The physical property such as mechanical strength of most hydrogels is not good enough. However, the properties of these hydrogels are easily controlled by changing the polymer concentration, molecular weight and cross-linker concentrations. It is found that the swelling degree increases with respect to the DMAAm concentration. The mechanical properties of transparent shape memory hydrogel were investigated by the tensile, the compression and the dynamic mechanical analysis. The Young’s modulus gradually decreases with the increase of hydrophilic components. On the other hand, higher Young’s modulus is observed by increasing the high hydrophobic concentration. It is also seen that the mechanical stress decreases with respect to the temperature, indicating the loss of Young’s modulus. The result of dynamic mechanical analysis designates that the water swollen sample has both the elastic and viscous properties

BMP2-induced gene profiling in dental epithelial cell line

Tooth development is regulated by epithelial-mesenchymal interactions and their reciprocal molecular signaling. Bone morphogenetic protein 2 (BMP2) is known as one of the inducers for tooth development. To analyze the molecular mechanisms of BMP2 on ameloblast differentiation (amelogenesis), we performed microarray analyses using rat dental epithelial cell line, HAT-7. After confirming that BMP2 could activate the canonical BMP-Smads signaling in HAT-7 cells, we analyzed the effects of BMP2 on 14,815 gene expressions and profiled them. Seventy-three genes were up-regulated and 28 genes were down-regulated by BMP2 treatment for 24 hours in HAT-7 cells. Functional classification revealed that 18% of up-regulated genes were ECM/adhesion molecules present in the enamel organ. Furthermore, we examined the expression of several differentiation markers in dental epithelial four cell-lineages including inner enamel epithelium (ameloblasts), stratum intermedium, stratum reticulum, and outer enamel epithelium. The results indicated that BMP2 might induce at least two different cell-lineage markers including a BMP antagonist expressed in HAT-7 cells, suggesting that BMP2 could accelerate amelogenesis via BMP signaling

Local application of Usag-1 siRNA can promote tooth regeneration in Runx2-deficient mice

Runt-related transcription factor 2 (Runx2)-deficient mice can be used to model congenital tooth agenesis in humans. Conversely, uterine sensitization-associated gene-1 (Usag-1)-deficient mice exhibit supernumerary tooth formation. Arrested tooth formation can be restored by crossing both knockout-mouse strains; however, it remains unclear whether topical inhibition of Usag-1 expression can enable the recovery of tooth formation in Runx2-deficient mice. Here, we tested whether inhibiting the topical expression of Usag-1 can reverse arrested tooth formation after Runx2 abrogation. The results showed that local application of Usag-1 Stealth small interfering RNA (siRNA) promoted tooth development following Runx2 siRNA-induced agenesis. Additionally, renal capsule transplantation of siRNA-loaded cationized, gelatin-treated mouse mandibles confirmed that cationized gelatin can serve as an effective drug-delivery system. We then performed renal capsule transplantation of wild-type and Runx2-knockout (KO) mouse mandibles, treated with Usag-1 siRNA, revealing that hindered tooth formation was rescued by Usag-1 knockdown. Furthermore, topically applied Usag-1 siRNA partially rescued arrested tooth development in Runx2-KO mice, demonstrating its potential for regenerating teeth in Runx2-deficient mice. Our findings have implications for developing topical treatments for congenital tooth agenesis

Hertwig\u27s epithelial root sheath cells contribute to formation of periodontal ligament through epithelial-mesenchymal transition by TGF-β.

In tooth root development, periodontal ligament (PDL) and cementum are formed by the coordination with the fragmentation of Hertwig\u27s epithelial root sheath (HERS) and the differentiation of dental follicle mesenchymal cells. However, the function of the dental epithelial cells after HERS fragmentation in the PDL is not fully understood. Here, we found that TGF-β regulated HERS fragmentation via epithelial-mesenchymal transition (EMT), and the fragmented epithelial cells differentiated into PDL fibroblastic cells with expressing of PDL extracellular matrix (ECM). In the histochemical analysis, TGF-β was expressed in odontoblast layer adjacent of HERS during root development. Periostin expression was detected around fragmented epithelial cells on the root surface, but not in HERS. In the experiment using an established mouse HERS cell line (HERS01a), TGF-β1 treatment decreased E-cadherin and relatively increased N-cadherin expression. TGF-β1 treatment in HERS01a induced further expression of important ECM proteins for acellular cementum and PDL development such as fibronectin and periostin. Taken together, activation of TGF-βsignaling induces HERS fragmentation through EMT and the fragmented HERS cells contribute to formation of PDL and acellular cementum through periostin and fibronectin expression.福岡歯科大学2016年

Induction of insulin-like growth factor 2 expression in a mesenchymal cell line co-cultured with an ameloblast cell line

Various growth factors have been implicated in the regulation of cell proliferation and differentiation during tooth development. It has been unclear if insulin-like growth factors (IGFs) participate in the epithelium–mesenchyme interactions of tooth development. We previously produced three-dimensional sandwich co-culture systems (SW) containing a collagen membrane that induce the differentiation of epithelial cells. In the present study, we used the SW system to analyze the expression of IGFs and IGFRs. We demonstrate that IGF2 expression in mesenchymal cells was increased by SW. IGF1R transduces a signal; however, IGF2R does not transduce a signal. Recombinant IGF2 induces IGF1R and IGF2R expression in epithelial cells. IGF1R expression is increased by SW; however, IGF2R expression did not increase by SW. Thus, IGF2 signaling works effectively in SW. These results suggest that IGF signaling acts through the collagen membrane on the interaction between the epithelium and mesenchyme. In SW, other cytokines may be suppressed to induce IGF2R induction. Our results suggest that IGF2 may play a role in tooth differentiation

Glyceraldehyde-3-phosphate Dehydrogenase Aggregates Accelerate Amyloid-β Amyloidogenesis in Alzheimer Disease

peer reviewedAlzheimer disease (AD) is a progressive neurodegenerative disorder characterized by loss of neurons and formation of pathological extracellular deposits induced by amyloid-β peptide (Aβ). Numerous studies have established Aβ amyloidogenesis as a hallmark of AD pathogenesis, particularly with respect to mitochondrial dysfunction. We have previously shown that glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forms amyloid-like aggregates upon exposure to oxidative stress and that these aggregates contribute to neuronal cell death. Here, we report that GAPDH aggregates accelerate Aβ amyloidogenesis and subsequent neuronal cell death both in vitro and in vivo. Co-incubation of Aβ40 with small amounts of GAPDH aggregates significantly enhanced Aβ40 amyloidogenesis, as assessed by in vitro thioflavin-T assays. Similarly, structural analyses using Congo red staining, circular dichroism, and atomic force microscopy revealed that GAPDH aggregates induced Aβ40 amyloidogenesis. In PC12 cells, GAPDH aggregates augmented Aβ40-induced cell death, concomitant with disruption of mitochondrial membrane potential. Furthermore, mice injected intracerebroventricularly with Aβ40 co-incubated with GAPDH aggregates exhibited Aβ40-induced pyramidal cell death and gliosis in the hippocampal CA3 region. These observations were accompanied by nuclear translocation of apoptosis-inducing factor and cytosolic release of cytochrome c from mitochondria. Finally, in the 3×Tg-AD mouse model of AD, GAPDH/Aβ co-aggregation and mitochondrial dysfunction were consistently detected in an age-dependent manner, and Aβ aggregate formation was attenuated by GAPDH siRNA treatment. Thus, this study suggests that GAPDH aggregates accelerate Aβ amyloidogenesis, subsequently leading to mitochondrial dysfunction and neuronal cell death in the pathogenesis of AD

エナメル質の横紋形成メカニズムの解明

エナメル質の基本構造をなすエナメル小柱には横紋が観察される.この横紋は概日リズムを刻んだ成長線のひとつとして知られているが,形成メカニズムについては様々な説がある.非脱灰凍結切片で基質形成期のエナメル基質を抗アメロゲニン抗体で免疫染色すると横紋様パターンを示した.この結果から,横紋は基質形成期のアメロゲニンのタンパク量に依存した石灰化パターンであり,アメロゲニンの発現は概日的に変動するのではないかと推測した.そこで,アメロゲニンの発現に周期があるか観察するために,アメロゲニンプロモーターの下流にルシフェラーゼを繋いだコンストラクト(pGL3-1730-luc)をラットエナメル芽細胞株HAT7に遺伝子導入してアメロゲニンの転写活性を計測したところ,一定の周期をもって変動することが認められた.次に,プロモーター領域のDeletion-mutant (pGL3-464, -74, -48-luc)を作製して周期性の制御に関わる領域を検索した結果, C/EBPαのbindingモチーフが有力な候補と考えられた.その転写はMsx2によって制御を受けることが知られていることから, Msx2の発現ベクターを用いた強制発現の影響を調べたところ,そのリズムが消失した.またMsx2の遺伝子欠損マウスには基質形成の横紋様パターンが見られなかった.以上の結果から, Msx2がアメロゲニンの発現周期に影響していることが推測され,さらにアメロゲニンの発現量の周期的変化によって生じた基質形成パターンが,横紋形成に関与していると考えられた.In enamel rods, periodic bands referred to as \u27cross-striations\u27 are observed, and known as incremental lines of circadian rhythm. Though it is considered that the cross-striation is involved in the biological circadian rhythm during the secretion of enamel matrix protein by ameloblasts, the developmental mechanism involved has not been examined in detail. By immunostaining amelogenin in fresh frozen sections of mouse lower incisor, we could observe fluorescent periodic bands in the enamel matrix, which were identical to the pattern of these cross-striations. Accordingly, we focused on the biological rhythm in the section of amelogenin. We examined amelogenin mRNA transcriptional activity in an ameloblast cell line (HAT7) by using real-time luminescence microscopy. The results showed that amelogenin mRNA transcriptional activity exhibited periodic rhythmicity and that Msx2 over-expression led to the disappearance of the periodic change. Further, in the lower incisors of Msx2-deficient mice, the periodic bands were not observed. Taken together, our findings suggest that the formation of cross-striations in the enamel rods was associated with the expression of amelogenin regulated by the transcriptional activity