nanotubes

纳米管
  • 文章类型: Journal Article
    DNA折纸纳米结构(DOs)是包括药物递送在内的应用的有前途的工具,生物传感,检测生物分子,探测染色质亚结构。将这些纳米设备靶向哺乳动物细胞核可以为探测提供有效的方法,可视化,并控制活细胞内的生物分子过程。我们提出了一种将DO递送到活细胞核中的方法。我们表明,这些DO在细胞培养基或细胞提取物中24小时内不会发生可检测的结构降解。为了将DOs传递到人类U2OS细胞的细胞核中,我们将30纳米DO纳米棒与针对核因子的抗体结合起来,特别是RNA聚合酶II(PolII)的最大亚基。我们发现DO在细胞中保持结构完整24小时,包括在细胞核内部。我们证明了电穿孔的抗PolII抗体缀合的DOs被搭载到细胞核中,并在细胞核内表现出亚扩散运动。我们的结果建立了具有核因子的接口DO作为将纳米设备递送到活细胞核的有效方法。
    DNA origami nanostructures (DOs) are promising tools for applications including drug delivery, biosensing, detecting biomolecules, and probing chromatin substructures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing, visualizing, and controlling biomolecular processes within live cells. We present an approach to deliver DOs into live-cell nuclei. We show that these DOs do not undergo detectable structural degradation in cell culture media or cell extracts for 24 hours. To deliver DOs into the nuclei of human U2OS cells, we conjugated 30-nanometer DO nanorods with an antibody raised against a nuclear factor, specifically the largest subunit of RNA polymerase II (Pol II). We find that DOs remain structurally intact in cells for 24 hours, including inside the nucleus. We demonstrate that electroporated anti-Pol II antibody-conjugated DOs are piggybacked into nuclei and exhibit subdiffusive motion inside the nucleus. Our results establish interfacing DOs with a nuclear factor as an effective method to deliver nanodevices into live-cell nuclei.
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  • 文章类型: Journal Article
    天然抗微生物肽(AMP)和酶(AME)是有希望的抗微生物耐药性的非抗生素候选物,但效率低且稳定性差。这里,我们开发了通过从头设计和肽组装模拟AMPs和AMEs作用模式的肽纳米酶。通过建模,通过将AMP和AME中的关键氨基酸与疏水性异亮氨酸组合以进行组装,提出了IHIHICI的最小构造块。实验验证表明,IHIHICI组装成具有乙酸调节的螺旋β-折叠纳米管,并具有Ni配位的磷脂酶C样和过氧化物酶样活性,表现出高的热稳定性和对酶降解的抗性。组装的纳米管表现出级联抗真菌作用,包括外甘露聚糖对接,墙壁破坏,脂质过氧化和随后的铁细胞死亡,在消毒垫上10分钟内协同杀死>90%的白色念珠菌。这些发现证明了开发具有多种抗微生物作用模式的材料的有效从头设计策略。
    Natural antimicrobial peptides (AMPs) and enzymes (AMEs) are promising non-antibiotic candidates against antimicrobial resistance but suffer from low efficiency and poor stability. Here, we develop peptide nanozymes which mimic the mode of action of AMPs and AMEs through de novo design and peptide assembly. Through modelling a minimal building block of IHIHICI is proposed by combining critical amino acids in AMPs and AMEs and hydrophobic isoleucine to conduct assembly. Experimental validations reveal that IHIHICI assemble into helical β-sheet nanotubes with acetate modulation and perform phospholipase C-like and peroxidase-like activities with Ni coordination, demonstrating high thermostability and resistance to enzymatic degradation. The assembled nanotubes demonstrate cascade antifungal actions including outer mannan docking, wall disruption, lipid peroxidation and subsequent ferroptotic death, synergistically killing >90% Candida albicans within 10 min on disinfection pad. These findings demonstrate an effective de novo design strategy for developing materials with multi-antimicrobial mode of actions.
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  • 文章类型: Journal Article
    背景:最近的研究证明了自噬在间充质干细胞(MSCs)功能和再生特性中的作用。应该解决自噬调节如何以及通过何种机制影响MSCs的并分泌相互作用。这里,研究了自噬在隧穿纳米管(TNTs)形成和同型线粒体捐献中的作用.
    方法:将MSCs与15µM二甲双胍(Met)和/或3µM3-甲基腺嘌呤(3-MA)孵育48小时。使用亮场和SEM图像评估TNTs的形成。使用流式细胞术分析监测线粒体密度和ΔΦ值。使用RT-PCR和蛋白质阵列,自噬之间的密切相互作用和共享介质,凋亡,和Wnt信号通路也被监测。使用气相色谱法评估总脂肪酸谱。
    结果:数据表明TNT长度和数量增加,以及诱导自噬后的其他细胞投射,而这些特征在3-MA处理的MSC中被钝化(p<0.05)。Western印迹显示Rab8和p-FAK在3-MA处理的MSCs中显著减少(p<0.05)。表明抑制TNT组装和囊泡运输。同样,与3-MA处理的MSCs相比,自噬刺激增加了自噬通量和线粒体膜完整性.尽管有这些发现,线粒体膜Miro1和2蛋白水平在自噬抑制/刺激后没有变化(p>0.05)。我们发现自噬的抑制/刺激可以影响蛋白质,与不同细胞生物活性相关的Wnt和凋亡信号通路相关的几种介质的转录水平。数据证实暴露于自噬刺激物的MSC中单和多不饱和/饱和脂肪酸比例的显著增加。
    结论:总之,自噬调节可能会影响同型线粒体捐赠所需的TNT形成。因此,自噬的调节为提高基于细胞的治疗效率创造了一个有希望的观点.
    BACKGROUND: Recent studies have proved the role of autophagy in mesenchymal stem cell (MSCs) function and regenerative properties. How and by which mechanism autophagy modulation can affect the juxtacrine interaction of MSCs should be addressed. Here, the role of autophagy was investigated in the formation of tunneling nanotubes (TNTs) and homotypic mitochondrial donation.
    METHODS: MSCs were incubated with 15 µM Metformin (Met) and/or 3 µM 3-methyladenine (3-MA) for 48 h. The formation of TNTs was assessed using bright-field and SEM images. The mitochondria density and ΔΨ values were monitored using flow cytometry analysis. Using RT-PCR and protein array, the close interaction and shared mediators between autophagy, apoptosis, and Wnt signaling pathways were also monitored. The total fatty acid profile was assessed using gas chromatography.
    RESULTS: Data indicated the increase of TNT length and number, along with other cell projections after the induction of autophagy while these features were blunted in 3-MA-treated MSCs (p < 0.05). Western blotting revealed the significant reduction of Rab8 and p-FAK in 3-MA-treated MSCs (p < 0.05), indicating the inhibition of TNT assembly and vesicle transport. Likewise, the stimulation of autophagy increased autophagic flux and mitochondrial membrane integrity compared to 3-MA-treated MSCs. Despite these findings, protein levels of mitochondrial membrane Miro1 and 2 were unchanged after autophagy inhibition/stimulation (p > 0.05). We found that the inhibition/stimulation of autophagy can affect the protein, and transcription levels of several mediators related to Wnt and apoptosis signaling pathways involved in different cell bioactivities. Data confirmed the profound increase of mono and polyunsaturated/saturated fatty acid ratio in MSCs exposed to autophagy stimulator.
    CONCLUSIONS: In summary, autophagy modulation could affect TNT formation which is required for homotypic mitochondrial donation. Thus, the modulation of autophagy creates a promising perspective to increase the efficiency of cell-based therapies.
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  • 文章类型: Journal Article
    无机层状化合物(2D材料),特别是过渡金属二硫属化物(TMDC),是近年来深入研究的焦点。在1991年发现碳纳米管(CNT)后不久,人们假设2D材料的纳米结构也可以折叠和接缝形成,从而纳米管(NT)。的确,在CNT之后不久报道了WS2的纳米管(和富勒烯样纳米颗粒)以及随后来自MoS2的纳米管(和富勒烯样纳米颗粒)。然而,TMDC纳米管比CNT受到的关注要少得多,直到最近,可能是因为它们不能容易地生产为具有明确定义的手性角度的单壁纳米管。尽管如此,最近几年来无机层状化合物的NTs已成为一个肥沃的研讨领域。在高温合成不同类型的TMDC纳米管方面取得了很大进展,以及它们的表征以及它们的性质和潜在应用的研究。他们的多墙结构被发现是一种祝福而不是诅咒,导致有趣的观察。这篇简明的小综述致力于TMDC纳米管研究的最新进展。在回顾了它们的合成和结构表征的进展后,他们对能量转换和储存研究领域的贡献,聚合物纳米复合材料,和独特的光电设备正在审查中。这些研究表明TMDC纳米管在各种技术中的许多潜在应用,简要讨论。
    Inorganic layered compounds (2D-materials), particularly transition metal dichalcogenide (TMDC), are the focus of intensive research in recent years. Shortly after the discovery of carbon nanotubes (CNTs) in 1991, it was hypothesized that nanostructures of 2D-materials can also fold and seam forming, thereby nanotubes (NTs). Indeed, nanotubes (and fullerene-like nanoparticles) of WS2 and subsequently from MoS2 were reported shortly after CNT. However, TMDC nanotubes received much less attention than CNT until recently, likely because they cannot be easily produced as single wall nanotubes with well-defined chiral angles. Nonetheless, NTs from inorganic layered compounds have become a fertile field of research in recent years. Much progress has been achieved in the high-temperature synthesis of TMDC nanotubes of different kinds, as well as their characterization and the study of their properties and potential applications. Their multiwall structure is found to be a blessing rather than a curse, leading to intriguing observations. This concise minireview is dedicated to the recent progress in the research of TMDC nanotubes. After reviewing the progress in their synthesis and structural characterization, their contributions to the research fields of energy conversion and storage, polymer nanocomposites, andunique optoelectronic devices are being reviewed. These studies suggest numerous potential applications for TMDC nanotubes in various technologies, which are briefly discussed.
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  • 文章类型: Journal Article
    在这项研究中,我们评估了通过各种施加电压序列通过阳极氧化制造的直径定制的TiO2纳米管层的药物释放行为:常规的恒定施加电位为20V(45nm)和60V(80nm),20/60V步进电位(50nm[两个直径]),和20-60V扫描电位(49nm[全锥形])(括号中的值表示纳米管层顶部的内管直径)。50nm(两个直径)和49nm(全锥形)样品的结构在纳米管层顶部的内径小于80nm样品的内径,而纳米管层底部的外径与80nm样品的尺寸几乎相同。80nm的样品,具有最大的纳米管直径和长度,展示了最大的爆发释放,其次是50nm(两个直径),49nm(全锥形),和45nm样品。较小的管顶部显著抑制了从50nm(两个直径)和49nm(全锥形)样品的初始突释药物量和释放速率。另一方面,对于50nm(两个直径)样品,观察到缓慢释放的药物量占总释放药物量的最大比例。因此,50nm(两个直径)实现了抑制的初始爆发释放和大的存储容量。因此,这项研究,第一次,应用具有调制直径(两个直径和全锥形)的TiO2纳米管层以实现具有定制药物释放特性的局部药物递送系统(LDDS)。
    In this study, we evaluated the drug release behavior of diameter customized TiO2 nanotube layers fabricated by anodization with various applied voltage sequences: conventional constant applied potentials of 20 V (45 nm) and 60 V (80 nm), a 20/60 V stepped potential (50 nm [two-diameter]), and a 20-60 V swept potential (49 nm [full-tapered]) (values in parentheses indicate the inner tube diameter at the top part of nanotube layers). The structures of the 50 nm (two-diameter) and 49 nm (full-tapered) samples had smaller inner diameters at the top part of nanotube layers than that of the 80 nm sample, while the outer diameters at the bottom part of nanotube layers were almost the same size as the 80 nm sample. The 80 nm sample, which had the largest nanotube diameter and length, exhibited the greatest burst release, followed by the 50 nm (two-diameter), 49 nm (full-tapered), and 45 nm samples. The initial burst released drug amounts and release rates from the 50 nm (two-diameter) and 49 nm (full-tapered) samples were significantly suppressed by the smaller tube top. On the other hand, the largest proportion of the slow released drug amount to the total released drug amount was observed for the 50 nm (two-diameter) sample. Thus, 50 nm (two-diameter) achieved suppressed initial burst release and large storage capacity. Therefore, this study has, for the first time, applied TiO2 nanotube layers with modulated diameters (two-diameter and full-tapered) to the realization of a localized drug delivery system (LDDS) with customized drug release properties.
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  • 文章类型: Journal Article
    受碳纳米管强烈光吸收的启发,我们提出了一种制造方法,涉及一维TiO2/Bi2S3QDs纳米管(TBNTs)具有可见红光可激发的光电性能。通过整合异质结的构造,量子限制效应,和形态学修饰,光电流达到9.22μA/cm2,是TiO2纳米管(TNTs)的66倍。然后,通过将红光响应的光电活性水凝胶敷料(TBCHA)将TBNTs嵌入到具有良好生物相容性的基于胶原/透明质酸的仿生细胞外基质水凝胶中,旨在促进伤口愈合和皮肤功能恢复。这种方法主要基于电刺激在调节神经功能和免疫反应中的公认重要性。严重烧伤常伴有上皮神经网络的广泛损伤,导致兴奋功能丧失和难以自发愈合,而常规敷料不足以解决神经神经支配的关键需求。此外,我们强调了TBCHA光电水凝胶促进神经末梢神经支配的非凡能力,促进皮肤亚结构的修复,并调节深度烧伤模型中的免疫反应。这种水凝胶不仅支持伤口闭合和胶原蛋白合成,而且促进血管重建,免疫调节,和神经恢复。这种基于光电的疗法为深度烧伤和功能性组织再生的全面修复提供了强大的解决方案。意义:我们探索了具有可见红光兴奋性和高光电转换性能的1DTiO2/Bi2S3纳米管的制备。通过集成异质结,量子吸收效应,和形态学修饰,TiO2/Bi2S3纳米管的光电流可达9.22μA/cm²,在625nm的光照下,这是TiO2纳米管的66倍。有效的红光兴奋性解决了由短波激发引起的生物安全性差和组织穿透率低的问题。此外,我们强调了TiO2/Bi2S3纳米管集成光电水凝胶在促进神经末梢神经支配和调节免疫反应方面的显着能力。这项工作提出了一种新兴的远程治疗策略,被动电刺激,为修复深度烧伤伤口提供了强大的助力。
    Inspired by the strong light absorption of carbon nanotubes, we propose a fabrication approach involving one-dimensional TiO2/Bi2S3 QDs nanotubes (TBNTs) with visible red-light excitable photoelectric properties. By integrating the construction of heterojunctions, quantum confinement effects, and morphological modifications, the photocurrent reached 9.22 μA/cm2 which is 66 times greater than that of TiO2 nanotubes (TNTs). Then, a red light-responsive photoelectroactive hydrogel dressing (TBCHA) was developed by embedding TBNTs into a collagen/hyaluronic acid-based biomimetic extracellular matrix hydrogel with good biocompatibility, aiming to promote wound healing and skin function restoration. This approach is primarily grounded in the recognized significance of electrical stimulation in modulating nerve function and immune responses. Severe burns are often accompanied by extensive damage to epithelial-neural networks, leading to a loss of excitatory function and difficulty in spontaneous healing, while conventional dressings inadequately address the critical need for nerve reinnervation. Furthermore, we highlight the remarkable ability of the TBCHA photoelectric hydrogel to promote the reinnervation of nerve endings, facilitate the repair of skin substructures, and modulate immune responses in a deep burn model. This hydrogel not only underpins wound closure and collagen synthesis but also advances vascular reformation, immune modulation, and neural restoration. This photoelectric-based therapy offers a robust solution for the comprehensive repair of deep burns and functional tissue regeneration. STATEMENT OF SIGNIFICANCE: We explore the fabrication of 1D TiO2/Bi2S3 nanotubes with visible red-light excitability and high photoelectric conversion properties. By integrating heterojunctions, quantum absorption effects, and morphological modifications, the photocurrent of TiO2/Bi2S3 nanotubes could reach 9.22 μA/cm², which is 66 times greater than that of TiO2 nanotubes under 625 nm illumination. The efficient red-light excitability solves the problem of poor biosafety and low tissue penetration caused by shortwave excitation. Furthermore, we highlight the remarkable ability of the TiO2/Bi2S3 nanotubes integrated photoelectric hydrogel in promoting the reinnervation of nerve endings and modulating immune responses. This work proposes an emerging therapeutic strategy of remote, passive electrical stimulation, offering a robust boost for repairing deep burn wounds.
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  • 文章类型: Journal Article
    通过水热合成开发了ZnO纳米棒无纺布(ZNRN),以促进预防呼吸道病原体的传播。通过响应面法提高了ZNRN的超疏水性和抗菌性能。合成的材料表现出显著的防水性,水接触角为163.9°,因此对大肠杆菌的抗菌率为91.8%(E.大肠杆菌)和79.75%的金黄色葡萄球菌(S.金黄色葡萄球菌)。这表明具有较薄肽聚糖的大肠杆菌可能比金黄色葡萄球菌更容易被杀死。这项研究确定了合成条件对抗菌效果的显着影响,全面的多变量分析阐明了潜在的相关性。此外,通过SEM和XRD分析对ZNRN的ZnO纳米棒结构进行了表征。它赋予超疏水性(从而防止细菌粘附到ZNRN表面)和抗菌能力(从而通过刺穿这些纳米棒破坏细胞)的性质。因此,希望将两个这样的特征对齐,以帮助支持个人防护设备的开发,这有助于避免呼吸道感染的传播。
    ZnO nanorod nonwoven fabrics (ZNRN) were developed through hydrothermal synthesis to facilitate the prevention of the transmission of respiratory pathogens. The superhydrophobicity and antibacterial properties of ZNRN were improved through the response surface methodology. The synthesized material exhibited significant water repellency, indicated by a water contact angle of 163.9°, and thus demonstrated antibacterial rates of 91.8% for Escherichia coli (E. coli) and 79.75% for Staphylococcus aureus (S. aureus). This indicated that E. coli with thinner peptidoglycan may be more easily killed than S. aureus. This study identified significant effects of synthesis conditions on the antibacterial effectiveness, with comprehensive multivariate analyses elucidating the underlying correlations. In addition, the ZnO nanorod structure of ZNRN was characterized through SEM and XRD analyses. It endows the properties of superhydrophobicity (thus preventing bacteria from adhering to the ZNRN surface) and antibacterial capacity (thus damaging cells through the puncturing of these nanorods). Consequently, the alignment of two such features is desired to help support the development of personal protective equipment, which assists in avoiding the spread of respiratory infections.
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  • 文章类型: Journal Article
    减少肾移植排斥反应的追求强调了发展非侵入性,精确的诊断技术。这些技术旨在检测抗体介导的排斥反应(ABMR)和T细胞介导的排斥反应(TCMR)。无症状,有潜在肾损害的风险。管理由ABMR和TCMR引起的拒绝的协议不同,和诊断传统上依赖于侵入性活检程序。因此,使用纳米传感芯片的会聚系统,拉曼光谱,并引入AI技术,以便于使用从没有重大异常的患者获得的血清样本进行诊断,ABMR,和肾移植后的TCMR。组织活检和Banff评分分析在组间进行验证,将同时获得的5μL血清添加到基于Au-ZnO纳米棒的表面增强拉曼散射传感芯片上,获得拉曼光谱信号。机器学习算法对主成分-线性判别分析和主成分-偏最小二乘判别分析的准确率分别为93.53%和98.82%,分别。胶原蛋白(肾损伤的指示),肌酐,和氨基酸衍生的信号(肾功能标志物)有助于这种准确性;然而,这种高准确性主要是由于该系统能够分析广谱的各种生物标志物.
    The quest to reduce kidney transplant rejection has emphasized the urgent requirement for the development of non-invasive, precise diagnostic technologies. These technologies aim to detect antibody-mediated rejection (ABMR) and T-cell-mediated rejection (TCMR), which are asymptomatic and pose a risk of potential kidney damage. The protocols for managing rejection caused by ABMR and TCMR differ, and diagnosis has traditionally relied on invasive biopsy procedures. Therefore, a convergence system using a nano-sensing chip, Raman spectroscopy, and AI technology was introduced to facilitate diagnosis using serum samples obtained from patients with no major abnormality, ABMR, and TCMR after kidney transplantation. Tissue biopsy and Banff score analysis were performed across the groups for validation, and 5 μL of serum obtained at the same time was added onto the Au-ZnO nanorod-based Surface-Enhanced Raman Scattering sensing chip to obtain Raman spectroscopy signals. The accuracy of machine learning algorithms for principal component-linear discriminant analysis and principal component-partial least squares discriminant analysis was 93.53% and 98.82%, respectively. The collagen (an indicative of kidney injury), creatinine, and amino acid-derived signals (markers of kidney function) contributed to this accuracy; however, the high accuracy was primarily due to the ability of the system to analyze a broad spectrum of various biomarkers.
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  • 文章类型: Journal Article
    层状或链状材料由于其有趣的物理性质而受到了重要的研究关注,当材料从块状(三维)变薄到薄的二维薄片或一维(1D)链形式时,这可能会发生巨大变化。具有A=Si或Ge和X=S或Se的化学计量AX2的材料形成特别吸引人的半导体类。例如,块状硅二硫族化合物(SiX2)由范德华力保持在一起的1D链组成。尽管这种结构配置有可能在一维极限内揭示有趣的物理现象,获得SiX2单链一直是具有挑战性的。我们在这里通过实验和理论检查低链数极限的SiX2材料。碳纳米管作为生长模板,稳定和保护结构,和原子分辨率扫描透射电子显微镜直接识别原子结构。对于SiX2观察到两种不同的链结构。还合成了SixGe1-xS2(1-y)Se2y四元合金链并对其进行了表征,展示了在原子链水平上可调的半导体特性。密度泛函理论计算表明,这些合金链的带隙可以通过成分工程进行广泛的调整。这项工作提供了在单链极限下合成和控制半导体成分以定制材料特性的可能性。
    Layered or chain materials have received significant research attention owing to their interesting physical properties, which can dramatically change when the material is thinned from bulk (three-dimensional) to thin two-dimensional sheet or one-dimensional (1D) chain form. Materials with the stoichiometry AX2 with A = Si or Ge and X = S or Se form an especially intriguing semiconducting class. For example, bulk silicon dichalcogenides (SiX2) consist of 1D chains held together by van der Waals forces. Although this structural configuration has the potential to reveal interesting physical phenomena within the 1D limit, obtaining SiX2 single chains has been challenging. We here examine experimentally and theoretically SiX2 materials in the low chain number limit. Carbon nanotubes serve as growth templates and stabilize and protect the structures, and atomic-resolution scanning transmission electron microscopy directly identifies the atomic structure. Two distinct chain structures are observed for SiX2. SixGe1-xS2(1-y)Se2y quaternary alloy chains are also synthesized and characterized, demonstrating tunable semiconducting properties at the atomic-chain level. Density functional theory calculations reveal that the band gap of these alloy chains can be widely tuned through composition engineering. This work offers the possibilities for synthesizing and controlling semiconductor compositions at the single-chain limit to tailor material properties.
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  • 文章类型: Journal Article
    骨整合是决定植入成功的最重要因素。通过阳极氧化制备的TiO2纳米管的表面改性在促进骨形成方面具有显著的优势。然而,这种现象背后的机制仍然难以理解。在这里,我们表明纳米形貌表现出开放和干净的纳米管结构和强亲水性,纳米形态显著促进了粘附,扩散,和干细胞的成骨分化。探索机制,我们发现纳米形态可以通过激活Piezo1和增加细胞内Ca2+来增强线粒体氧化磷酸化(OxPhos)。OxPhos的增加可以显着提高细胞质中乙酰辅酶A的水平,但不会显着提高细胞核中乙酰辅酶A的水平。这有利于β-catenin的乙酰化和稳定性,并最终促进成骨。本研究为纳米形态学研究干细胞成骨的调控机制提供了新的解释。
    Osseointegration is the most important factor determining implant success. The surface modification of TiO2 nanotubes prepared by anodic oxidation has remarkable advantages in promoting bone formation. However, the mechanism behind this phenomenon is still unintelligible. Here we show that the nanomorphology exhibited open and clean nanotube structure and strong hydrophilicity, and the nanomorphology significantly facilitated the adhesion, proliferation, and osteogenesis differentiation of stem cells. Exploring the mechanism, we found that the nanomorphology can enhance mitochondrial oxidative phosphorylation (OxPhos) by activating Piezo1 and increasing intracellular Ca2+. The increase in OxPhos can significantly uplift the level of acetyl-CoA in the cytoplasm but not significantly raise the level of acetyl-CoA in the nucleus, which was beneficial for the acetylation and stability of β-catenin and ultimately promoted osteogenesis. This study provides a new interpretation for the regulatory mechanism of stem cell osteogenesis by nanomorphology.
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