Effect of NAMPT on the proliferation and apoptosis in odontoblast-like MDPC-23 cell.

This study aimed to evaluate the physiological role of NAMPT associated with MDPC-23 odontoblast cell proliferation. Cell viability was measured using the (DAPI) staining, caspase activation analysis and immunoblotting were performed. Visfatin promoted MDPC-23 odontoblast cell growth in a dose-dependent manner. Furthermore, the up-regulation of Visfatin promoted odontogenic differentiation and accelerated mineralization through an increase in representative odontoblastic biomarkers in MDPC-23 cells. However, FK-866 cell growth in a dose-dependent manner induced nuclear condensation and fragmentation. FK-866-treated cells showed H&E staining and increased apoptosis compared to control cells. The expression of anti-apoptotic factors components of the mitochondria-dependent intrinsic apoptotic pathway significantly decreased following FK-866 treatment. The expression of pro-apoptotic increased upon FK-866 treatment. In addition, FK-866 activated caspase-3 and PARP to induce cell death. In addition, after treating FK-866 for 72 h, the 3/7 activity of MDPC-23 cells increased in a concentration-dependent manner, and the IHC results also confirmed that Caspase-3 increased in a concentration-dependent. Therefore, the presence or absence of NAMPT expression in dentin cells was closely related to cell proliferation and formation of extracellular substrates.

Metformin Attenuates Manganese-Induced Oxidative Stress in N27-A Dopaminergic Neuronal Cells.

Metformin is an anti-diabetic drug that exerts protective effects against neurodegenerative diseases. In this study, we investigated the protective effects of metformin against manganese (Mn)-induced cytotoxicity associated with Parkinson's disease-like symptoms in N27-A dopaminergic (DA) cells. Metformin (0.1-1 mM) suppressed Mn (0.4 mM)-induced cell death in a concentration-dependent manner. Metformin pretreatment effectively suppressed the Mn-mediated increase in the levels of oxidative stress markers, such as reactive oxygen species (ROS) and thiobarbituric acid reactive substances. Moreover, metformin restored the levels of the antioxidants, superoxide dismutase, intracellular glutathione, and glutathione peroxidase, which were reduced by Mn. Metformin (0.5 mM) significantly attenuated the decrease in sirtuin-1 (SIRT1) and peroxisome proliferator activated receptor gamma coactivator-1 alpha levels, which were increased by Mn (0.4 mM). In addition, metformin inhibited the expression of microRNA-34a, which directly targeted SIRT1. Metformin also inhibited the loss of Mn-induced mitochondrial membrane potential (ΔΨm) and activation of the apoptosis marker, caspase-3. Furthermore, metformin-mediated inhibition of ROS generation and caspase-3 activation, recovery of ΔΨm, and restoration of cell viability were partially reversed by the SIRT1 inhibitor, Ex527. These results suggest that metformin may protects against Mn-induced DA neuronal cell death mediated by oxidative stress and mitochondrial dysfunction possibly via the regulation of SIRT1 pathway.

The Relationship Between Low Back Pain and Sagittal Spinal Alignment and Back Muscle Mass in Korean Fishery Workers.

OBJECTIVE: To investigate the relationship between low back pain (LBP) and sagittal spino-pelvic parameters along with the relationship between LBP and back muscle mass in Korean male and female fishery workers. METHODS: This retrospective study included a total of 146 subjects who underwent Fishermen's health survey conducted between June 2018 and August 2020. LBP was evaluated through visual analogue scale (VAS) and Oswestry Disability Index (ODI). Sagittal spino-pelvic parameters were measured from whole spine standing X-rays. Back muscle cross-sectional areas were identified through lumbar spine magnetic resonance imaging and body composition was analyzed through bioelectrical impedance analysis. RESULTS: The study included 75 males and 71 females, with an average age of 58.33 years for males and 56.45 years for females. Female subjects exhibited higher VAS and ODI scores, larger pelvic tilt (PT) and trunk fat mass and smaller trunk muscle mass compared to males. In female, ODI positively correlated with sagittal vertical axis (SVA) and PT. No significant correlations were found between ODI scores and body composition in either sex. The high ODI group showed greater SVA, PT, and pelvic incidence-lumbar lordosis and higher trunk fat/muscle ratio. Psoas muscle mass, total and skeletal muscle mass and trunk muscle mass of that group were smaller than those of low ODI group. CONCLUSION: Spino-pelvic parameters and back muscle mass were associated with ODI and there were differences between Korean male and female fishery workers.

7α,25-Dihydroxycholesterol-Induced Oxiapoptophagic Chondrocyte Death via the Modulation of p53-Akt-mTOR Axis in Osteoarthritis Pathogenesis.

This study aimed to exploring the pathophysiological mechanism of 7α,25-dihydroxycholesterol (7α,25-DHC) in osteoarthritis (OA) pathogenesis. 7α,25-DHC accelerated the proteoglycan loss in ex vivo organ-cultured articular cartilage explant. It was mediated by the decreasing extracellular matrix major components, including aggrecan and type II collagen, and the increasing expression and activation of degenerative enzymes, including matrix metalloproteinase (MMP)-3 and -13, in chondrocytes cultured with 7α,25-DHC. Furthermore, 7α,25-DHC promoted caspase dependent chondrocytes death via extrinsic and intrinsic pathways of apoptosis. Moreover, 7α,25-DHC upregulated the expression of inflammatory factors, including inducible nitric oxide synthase, cyclooxygenase-2, nitric oxide, and prostaglandin E2, via the production of reactive oxygen species via increase of oxidative stress in chondrocytes. In addition, 7α,25-DHC upregulated the expression of autophagy biomarker, including beclin-1 and microtubule-associated protein 1A/1B-light chain 3 via the modulation of p53-Akt-mTOR axis in chondrocytes. The expression of CYP7B1, caspase-3, and beclin-1 was elevated in the degenerative articular cartilage of mouse knee joint with OA. Taken together, our findings suggest that 7α,25-DHC is a pathophysiological risk factor of OA pathogenesis that is mediated a chondrocytes death via oxiapoptophagy, which is a mixed mode of apoptosis, oxidative stress, and autophagy.

The Ethanol Extracts of Osmanthus fragrans Leaves Ameliorate the Bone Loss via the Inhibition of Osteoclastogenesis in Osteoporosis.

The aim of this study was to evaluate the anti-osteoporosis effects of Osmanthus fragrans leaf ethanol extract (OFLEE) in bone marrow-derived macrophages (BMM) and animals with osteoporosis. OFLEE not only suppressed tartrate-resistant acid phosphatase (TRAP)-positive cells with multiple nuclei but also decreased TRAP activity in BMM treated with macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL). The formation of F-actin rings and the expression and activation of matrix metalloproteinases were decreased by OFLEE in BMM treated with M-CSF and RANKL. OFLEE suppressed M-CSF- and RANKL-induced osteoclastogenesis by inhibiting NF-κB phosphorylation, tumor necrosis factor receptor-associated factor 6, c-fos, the nuclear factor of activated T-cells, cytoplasmic 1, and cathepsin K in BMM. OFLEE downregulated reactive oxygen species, cyclooxygenase-2, inducible nitric oxide synthase, prostaglandin E2, tumor necrosis factor α, interleukin (IL)-1ß, IL-6, IL-17, and RANKL in BMM treated with M-CSF and RANKL. Oral administration of OFLEE suppressed osteoporotic bone loss without hepatotoxicity in ovariectomy-induced osteoporosis animals. Our findings suggest that OFLEE, with anti-inflammatory effects, prevents osteoporotic bone loss through the suppression of osteoclastic differentiation in BMM and animals with osteoporosis.

25-Hydroxycholesterol-induced Osteoblast Oxiapoptophagy Is Involved in the Pathophysiological Process of Osteoporosis.

BACKGROUND/AIM: 25-hydroxycholesterol (25-HC) plays important roles in lipid metabolism, inflammatory responses, and apoptosis, but its pathophysiological association with osteoporosis (OP) has not been verified in osteoblasts. Hence, we studied the pathophysiological linkage and underlying cellular mechanisms of 25-HC in human osteoblast-like MG-63 cells and an ovariectomy-induced osteoporotic mouse model. MATERIALS AND METHODS: To investigate the pathophysiological linkage between 25-HC-induced osteoblast oxiapoptophagy and OP, 25-HC ELISA assay, MTT assay, cell live/dead staining, hematoxylin and eosin staining, DAPI staining, flow cytometry analysis, western blot, caspase-3 staining, reactive oxygen species (ROS) assay, autophagy staining, immunocytochemistry, Micro-CT image analysis and immunocytochemistry were performed in MG-63 cells and ovariectomy-induced OP animals. RESULTS: The expression of cholesterol-25-hydroxylase (CH25H), an enzyme catalyzing the conversion of cholesterol to 25-HC, and the production of 25-HC were increased by lipopolysaccharide in MG-63 cells. Cytotoxicity was increased by 25-HC in MG-63 cells. Apoptosis with condensed chromatin and altered morphology was induced by 25-HC through cleavage of caspases-8, -9, and -3 in MG-63 cells. 25-HC induced oxidative stress in MG-63 cells via elevation of ROS production, cyclooxygenase-2, and inducible nitric oxide synthase. Furthermore, the expression of autophagy biomarkers, including beclin-1 and microtubule-associated protein 1A/1B-light chain 3, was elevated by 25-HC in MG-63 cells. In addition, p53 expression was increased, whereas Akt phosphorylation was suppressed in 25-HC-incubated MG-63 cells. The expression of CH25H, cleaved caspase-3, and beclin-1 were up-regulated in the femoral bone of ovariectomy-induced mouse osteoporotic animals. CONCLUSION: 25-HC plays a role in OP via the induction of oxiapoptophagic osteoblast death.

25-Hydroxycholesterol induces odontoclastic differentiation through RANK-RANKL upregulation and NF-κB activation in odontoblast-like MDPC-23 cells: An in vitro study.

AIM: The physiological effects and cellular mechanism of 25-hydroxycholesterol (25-HC), which is an oxysterol synthesized from cholesterol by cholesterol-25-hydroxylase (CH25H) expressed under inflammatory conditions, are still largely unknown during odontoclastogenesis. This study aimed to evaluate 25-HC-induced odontoclastogenesis and its cellular mechanisms in odontoblast-like MDPC-23 cells. METHODOLOGY: To investigate 25-HC-induced odontoclastogenesis of MDPC-23 cells and its cellular mechanism, haemotoxylin and eosin staining, tartrate-resistant acid phosphatase (TRAP) staining, dentine resorption assay, zymography, reactive oxygen species (ROS) detection, immunocytochemistry, and nuclear translocation were performed. The experimental values are presented as mean ± standard deviation and were compared using analysis of variance, followed by post hoc multiple comparisons (Tukey's test) using SPSS software version 22 (IBM Corp.). A p-value <.05 was considered statistically significant. RESULTS: Lipopolysaccharide or receptor activator of nuclear factor-κB ligand (RANKL) induced the synthesis of 25-HC via the expression of CH25H in MDPC-23 cells (p < .01). Multinucleated giant cells with morphological characteristics and TRAP activity of the odontoclast were increased by 25-HC in MDPC-23 cells (p < .01). Moreover, 25-HC increased dentine resorption through the expression and activity of matrix metalloproteinases in MDPC-23 cells. It not only increased the expression of odontoclastogenic biomarkers but also translocated cytosolic nuclear factor-κB (NF-κB) to the nucleus in MDPC-23 cells. Additionally, 25-HC not only increased the production of ROS (p < .01), expression of inflammatory mediators (p < .01), pro-inflammatory cytokines, receptor activator of NF-κB (RANK), and RANKL but also suppressed the expression of osteoprotegerin (OPG) in MDPC-23 cells. In contrast, CDDO-Me, a chemical NF-κB inhibitor, decreased TRAP activity (p < .01) and downregulated the expression of the odontoclastogenic biomarkers, including RANK and RANKL, in MDPC-23 cells. CONCLUSION: 25-HC induced odontoclastogenesis by modulating the RANK-RANKL-OPG axis via NF-κB activation in MDPC-23 cells. Therefore, these findings provide that 25-HC derived from cholesterol metabolism may be involved in the pathophysiological etiological factors of internal tooth resorption.

Arctigenin induces caspase-dependent apoptosis in FaDu human pharyngeal carcinoma cells.

The present study was carried out to investigate the effect of Arctigenin on cell growth and the mechanism of cell death elicited by Arctigenin were examined in FaDu human pharyngeal carcinoma cells. To determine the apoptotic activity of Arctigenin in FaDu human pharyngeal carcinoma cells, cell viability assay, DAPI staining, caspase activation analysis, and immunoblotting were performed. Arctigenin inhibited the growth of cells in a dose-dependent manner and induced nuclear condensation and fragmentation. Arctigenin-treated cells showed caspase-3/7 activation and increased apoptosis versus control cells. FasL, a death ligand associated with extrinsic apoptotic signaling pathways, was up-regulated by Arctigenin treatment. Moreover, caspase-8, a part of the extrinsic apoptotic pathway, was activated by Arctigenin treatments. Expressions of anti-apoptotic factors such as Bcl-2 and Bcl-xL, components of the mitochondria-dependent intrinsic apoptosis pathway, significantly decreased following Arctigenin treatment. The expressions of pro-apoptotic factors such as BAX, BAD and caspase-9, and tumor suppressor -53 increased by Arctigenin treatments. In addition, Arctigenin activated caspase-3 and poly (ADP-ribose) polymerase (PARP) induced cell death. Arctigenin also inhibited the proliferation of FaDu cells by the suppression of p38, NF-κB, and Akt signaling pathways. These results suggest that Arctigenin may inhibit cell proliferation and induce apoptotic cell death in FaDu human pharyngeal carcinoma cells through both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway.

GPR183 Regulates 7α,25-Dihydroxycholesterol-Induced Oxiapoptophagy in L929 Mouse Fibroblast Cell.

7α,25-dihydroxycholesterol (7α,25-DHC) is an oxysterol synthesized from 25-hydroxycholesterol by cytochrome P450 family 7 subfamily B member 1 (CYP7B1) and is a monooxygenase (oxysterol-7α-hydroxylase) expressed under inflammatory conditions in various cell types. In this study, we verified that 7α,25-DHC-induced oxiapoptophagy is mediated by apoptosis, oxidative stress, and autophagy in L929 mouse fibroblasts. MTT assays and live/dead cell staining revealed that cytotoxicity was increased by 7α,25-DHC in L929 cells. Consequentially, cells with condensed chromatin and altered morphology were enhanced in L929 cells incubated with 7α,25-DHC for 48 h. Furthermore, apoptotic population was increased by 7α,25-DHC exposure through the cascade activation of caspase-9, caspase-3, and poly (ADP-ribose) polymerase in the intrinsic pathway of apoptosis in these cells. 7α,25-DHC upregulated reactive oxygen species (ROS) in L929 cells. Expression of autophagy biomarkers, including beclin-1 and LC3, was significantly increased by 7α,25-DHC treatment in L929 cells. 7α,25-DHC inhibits the phosphorylation of Akt associated with autophagy and increases p53 expression in L929 cells. In addition, inhibition of G-protein-coupled receptor 183 (GPR183), a receptor of 7α,25-DHC, using GPR183 specific antagonist NIBR189 suppressed 7α,25-DHC-induced apoptosis, ROS production, and autophagy in L929 cells. Collectively, GPR183 regulates 7α,25-DHC-induced oxiapoptophagy in L929 cells.

Demethoxycurcumin induces apoptosis via inhibition of NF-κB pathway in FaDu human head and neck squamous cell carcinoma.

Background: Demethoxycurcumin (DMC) is a curcumin analog with antitumor properties. However, its effects have not been investigated in human head and neck squamous cell carcinoma (HNSCC). The aim of the present study was to verify the antitumor effect and cellular signaling pathways of DMC in FaDu HNSCC cells. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell Live/Dead staining, hematoxylin and eosin staining, DAPI staining, FACS, western blotting, caspase-3 activity assay, and nuclear translocation were performed to verify apoptosis and the cellular signaling pathway of DMC in FaDu cells. Results: DMC increased FaDu cell death, with cells presenting altered morphology and condensed nuclei. DMC increased significantly the apoptotic population of FaDu cells. Sequentially, DMC increased the expression of cleaved caspase-3 and PARP through the up-regulation of pro-apoptotic factors such as FasL, cleaved caspase-8, Bax, Bad, and cleaved caspase-9 and the suppression of anti-apoptotic factors including Bcl-xL and Bcl-2 in FaDu cells. Furthermore, DMC not only suppressed the phosphorylation of NF-κB, but also inhibited the translocation of NF-κB from cytosol to nucleus of FaDu cells. Conclusions: Present study demonstrates that DMC-induced cell death is mediated caspase-dependently by death receptor-mediated extrinsic and mitochondria-dependent intrinsic apoptosis through the inhibition of NF-κB translocation from the cytosol to the nucleus of FaDu cells. DMC is a curcuminoid with antitumor properties that modulates the NF-κB cellular signaling pathway in FaDu cells. Taken together, this study suggests that DMC has a considerable chemotherapeutic potential for HNSCC.

Nicotinamide phosphoribosyltransferase regulates the cell differentiation and mineralization in cultured odontoblasts.

The aim of the present study was to investigate the physiological role of nicotinamide phosphoribosyltransferase (NAMPT) associated with odontogenic differentiation during tooth development in mice. Mouse dental papilla cell-23 (MDPC- 23) cells cultured in differentiation media were stimulated with the specific NAMPT inhibitor, FK866, and Visfatin (NAMPT) for up to 10 days. The cells were evaluated after 0, 4, 7, and 10 days. Cell viability was measured using the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay. The mineralization assay was performed by staining MDPC-23 cells with Alizarin Red S solution. After cultivation, MDPC-23 cells were harvested for quantitative PCR or Western blotting. Analysis of variance was performed using StatView 5.0 software (SAS Institute Inc., Cary, NC, USA). Statistical significance was set at p < 0.05. The expression of NAMPT increased during the differentiation of murine odontoblast-like MDPC-23 cells. Furthermore, the up-regulation of NAMPT promoted odontogenic differentiation and accelerated mineralization through an increase in representative odontoblastic biomarkers, such as dentin sialophosphoprotein, dentin matrix protein-1, and alkaline phosphatase in MDPC-23 cells. However, treatment of the cells with the NAMPT inhibitor, FK866, attenuated odontogenic differentiation, as evidenced by the suppression of odontoblastic biomarkers. These data indicate that NAMPT regulated odontoblastic differentiation through the regulation of odontoblastic biomarkers. The increase in NAMPT expression in odontoblasts was closely related to the formation of the extracellular matrix and dentin via the Runx signaling pathway. Therefore, these data suggest that NAMPT is a critical regulator of odontoblast differentiation during tooth development.

Acteoside Counteracts Interleukin-1ß-Induced Catabolic Processes through the Modulation of Mitogen-Activated Protein Kinases and the NFκB Cellular Signaling Pathway.

Osteoarthritis (OA) is the most common degenerative joint disease with chronic joint pain caused by progressive degeneration of articular cartilage at synovial joints. Acteoside, a caffeoylphenylethanoid glycoside, has various biological activities such as antimicrobial, anti-inflammatory, anticancer, antioxidative, cytoprotective, and neuroprotective effect. Further, oral administration of acteoside at high dosage does not cause genotoxicity. Therefore, the aim of present study is to verify the anticatabolic effects of acteoside against osteoarthritis and its anticatabolic signaling pathway. Acteoside did not decrease the viabilities of mouse fibroblast L929 cells used as normal cells and primary rat chondrocytes. Acteoside counteracted the IL-1ß-induced proteoglycan loss in the chondrocytes and articular cartilage through suppressing the expression and activation of cartilage-degrading enzyme such as matrix metalloproteinase- (MMP-) 13, MMP-1, and MMP-3. Furthermore, acteoside suppressed the expression of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, nitric oxide, and prostaglandin E2 in the primary rat chondrocytes treated with IL-1ß. Subsequently, the expression of proinflammatory cytokines was decreased by acteoside in the primary rat chondrocytes treated with IL-1ß. Moreover, acteoside suppressed not only the phosphorylation of mitogen-activated protein kinases in primary rat chondrocytes treated with IL-1ß but also the translocation of NFκB from the cytosol to the nucleus through suppression of its phosphorylation. Oral administration of 5 and 10 mg/kg acteoside attenuated the progressive degeneration of articular cartilage in the osteoarthritic mouse model generated by destabilization of the medial meniscus. Our findings indicate that acteoside is a promising potential anticatabolic agent or supplement to attenuate or prevent progressive degeneration of articular cartilage.

Neuroprotective Effect of Cannabidiol Against Hydrogen Peroxide in Hippocampal Neuron Culture.

Introduction: Reports on the neurotoxic and neuroprotective effects of cannabidiol (CBD) have not been in complete accord, showing different and somewhat contradictory results depending upon the brain cell types and experimental conditions employed. This work systematically examines the neuroprotective capability of CBD against oxidative stress (i.e., hydrogen peroxide [H2O2]) as well as its toxicity profile in the in vitro culture platform of primary hippocampal neurons. Materials and Methods: The low cell-density (100 neurons per mm2) culture was used for analyzing the viability and morphology of neurons at a single-cell level with a confocal laser-scanning microscope (CLSM). Primary neurons were obtained from the hippocampal tissues of embryonic day-18 (E18) Sprague-Dawley rat pups and treated with CBD (0.1-100 µM) and/or H2O2 (0.1-50 µM) at 1 DIV (days in vitro). Results: The lethal concentration 50 (LC50) value (the concentration causing 50% cell death) of CBD was calculated to be 9.85 µM after 24 h of incubation, and that of H2O2 was 2.46 µM under the same conditions. The neuroprotection ratio of CBD against H2O2 ([H2O2]=10 µM) was 2.40 with 5 µM of CBD, increasing the cell viability to 57% from 24%. The CLSM analysis suggested that the cell-death mechanisms were different for CBD and H2O2, and CBD did not completely rescue the morphological alterations of primary hippocampal neurons caused by H2O2, such as neurite degeneration, at least in the in vitro neuron culture. Conclusion: Although CBD showed both neurotoxic and neuroprotective effects on hippocampal neurons in the in vitro setting, the use of low-concentrated (i.e., 5 µM) CBD, not causing toxic effects on the neurons, significantly rescued the neurons from the oxidative stress (H2O2), confirming its neuroprotection capability.

25-Hydroxycholesterol-Induced Oxiapoptophagy in L929 Mouse Fibroblast Cell Line.

25-hydroxycholesterol (25-HC) is an oxysterol synthesized from cholesterol by cholesterol-25-hydroxylase during cholesterol metabolism. The aim of this study was to verify whether 25-HC induces oxiapoptophagy in fibroblasts. 25-HC not only decreased the survival of L929 cells, but also increased the number of cells with condensed chromatin and altered morphology. Fluorescence-activated cell sorting results showed that there was a dose-dependent increase in the apoptotic populations of L929 cells upon treatment with 25-HC. 25-HC-induced apoptotic cell death was mediated by the death receptor-dependent extrinsic and mitochondria-dependent intrinsic apoptosis pathway, through the cascade activation of caspases including caspase-8, -9, and -3 in L929 cells. There was an increase in the levels of reactive oxygen species and inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, nitric oxide, and prostaglandin E2 in L929 cells treated with 25-HC. Moreover, 25-HC caused an increase in the expression of beclin-1 and microtubule-associated protein 1A/1B-light chain 3, an autophagy biomarker, in L929 cells. There was a significant decrease in the phosphorylation of protein kinase B (Akt) in L929 cells treated with 25-HC. Taken together, 25-HC induced oxiapoptophagy through the modulation of Akt and p53 cellular signaling pathways in L929 cells.

Neuronal Migration on Silicon Microcone Arrays with Different Pitches.

Neuronal migration is a complicated but fundamental process for proper construction and functioning of neural circuits in the brain. Many in vivo studies have suggested the involvement of environmental physical features of a neuron in its migration, but little effort has been made for the in vitro demonstration of topography-driven neuronal migration. This work investigates migratory behaviors of primary hippocampal neurons on a silicon microcone (SiMC) array that presents 14 different pitch domains (pitch: 2.5-7.3 µm). Neuronal migration becomes the maximum at the pitch of around 3 µm, with an upper migration threshold of about 4 µm. Immunocytochemical studies indicate that the speed and direction of migration, as well as its probability of occurrence, are correlated with the morphology of the neuron, which is dictated by the pitch and shape of underlying SiMC structures. In addition to the effects on neuronal migration, the real-time imaging of migrating neurons on the topographical substrate reveals new in vitro modes of neuronal migration, which have not been observed on the conventional flat culture plate, but been suggested by in vivo studies.

Fabrication and Characterization of Neurocompatible Ulvan-Based Layer-by-Layer Films.

Construction of extracellular matrix-mimetic nanofilms has considerable potential in biomedical and nanomedicinal fields. In this work, we fabricated neurocompatible layer-by-layer (LbL) films based on ulvan (ULV), a highly sulfated polysaccharide having compositional similarity to glycosaminoglycans that play important functional roles in the brain. ULV was durably assembled as a film with chitosan, another marine-derived polysaccharide, and the film enabled the stable adhesion of primary hippocampal neurons with high viability, comparable to the conventional poly-d-lysine surface. Notably, the ULV-based LbL films accelerated neurite outgrowth and selectively suppressed the adhesion of astrocytes, highlighting its potential as an advanced platform for neural implants and devices.

Neuro-taxis: Neuronal movement in gradients of chemical and physical environments.

Environmental chemical and physical cues dynamically interact with migrating neurons and sprouting axons, and in particular, the gradients of environmental cues are regarded as one of the factors intimately involved in the neuronal movement. Since a growth cone was first described by Cajal, more than one century ago, chemical gradients have been suggested as one of the mechanisms by which the neurons determine proper paths and destinations. However, the gradients of physical cues, such as stiffness and topography, which also interact constantly with the neurons and their axons as a component of the extracellular environments, have rarely been noted regarding the guidance of neurons, despite their gradually increasingly reported influences in the case of nonneuronal-cell migration. In this review, we discuss chemical (i.e., chemo- and hapto-) and physical (i.e., duro-) taxis phenomena on the movement of neurons including axonal elongation. In addition, we suggest topotaxis, the most recently proposed physical-taxis phenomenon, as another potential mechanism in the neuronal movement, based on the reports of neuronal recognition of and responses to nanotopography.

Astrocyte-Encapsulated Hydrogel Microfibers Enhance Neuronal Circuit Generation.

Astrocytes, the most representative glial cells in the brain, play a multitude of crucial functions for proper neuronal development and synaptic-network formation, including neuroprotection as well as physical and chemical support. However, little attention has been paid, in the neuroregenerative medicine and related fields, to the cytoprotective incorporation of astrocytes into neuron-culture scaffolds and full-fledged functional utilization of encapsulated astrocytes for controlled neuronal development. In this article, a 3D neurosupportive culture system for enhanced induction of neuronal circuit generation is reported, where astrocytes are confined in hydrogel microfibers and protected from the outside. The astrocyte-encapsulated microfibers significantly accelerate the neurite outgrowth and guide its directionality, and enhance the synaptic formation, without any physical contact with the neurons. This astrocyte-laden system provides a pivotal culture scaffold for advanced development of cell-based therapeutics for neural injuries, such as spinal cord injury.

Multiplexed Metabolic Labeling of Glycoconjugates in Polarized Primary Cerebral Cortical Neurons.

The spatial distribution of cell-surface glycoconjugates in the brain changes continuously, reflecting neurophysiology especially in the developing phase, but their functions and fates mostly remain unexplored. Their spatiotemporal distribution is particularly important in polarized neuronal cells, such as cerebral cortical neurons composed of a soma and neurites. In this work, we dually labeled sialic acid (Sia5Ac) and N-acetylgalactosamine/glucosamine (GalNAc/GlcNAc) by a neurocompatible strategy of metabolic glycan labeling, metabolism-by-tissues (MbT), and obtained the multiplexed information on their spatiotemporal distribution on polarized cortical neurons. The analyses showed the preferentially distinct distribution of each saccharide set at the late developmental stage after randomized, heterogeneous distribution at the early stage, suggesting that Sia5Ac and GalNAc/GlcNAc are translocated anisotropically during neuronal development.

Nanotopography-Promoted Formation of Axon Collateral Branches of Hippocampal Neurons.

Axon collateral branches, as a key structural motif of neurons, allow neurons to integrate information from highly interconnected, divergent networks by establishing terminal boutons. Although physical cues are generally known to have a comprehensive range of effects on neuronal development, their involvement in axonal branching remains elusive. Herein, it is demonstrated that the nanopillar arrays significantly increase the number of axon collateral branches and also promote their growth. Immunostaining and biochemical analyses indicate that the physical interactions between the nanopillars and the neurons give rise to lateral filopodia at the axon shaft via cytoskeletal changes, leading to the formation of axonal branches. This report, demonstrates that nanotopography regulates axonal branching, and provides a guideline for the design of sophisticated neuron-based devices and scaffolds for neuro-engineering.