Microneedles (MNs) are micron-sized needles that can penetrate the stratum corneum, enabling the non-invasive and painless administration of drugs and vaccines. In this work, fabrication conditions for high-aspect-ratio MNs by the photopolymerization of polyethylene glycol diacrylate (PEGDA) were investigated. Ultraviolet (UV) light was used to crosslink photocurable prepolymers in specific areas defined by a photomask. The aspect ratio of solidified MNs is too small to penetrate the stratum corneum if the degree of polymerization is insufficient. However, if the degree of polymerization is too high, a film is formed between the MNs by solidification of an undesired area owing to the scattering effect, reducing needle height. The influence of prepolymer molecular weight and the degree of UV absorption by the photoinitiator (PI) were studied to optimize the conditions for obtaining high-aspect-ratio MNs. Additionally, the effect of spacing ratio on high-aspect-ratio MNs without film formation has been discussed. A penetration test was conducted with porcine skin to analyze the effect of mechanical properties of MN. This study could guide the fabrication of MNs by the photopolymerization of biocompatible polymers with a photomask. Fabrication conditions for high-aspect-ratio MNs by the photopolymerization of PEGDA were investigated. The influence of prepolymer molecular weight and the degree of UV absorption by the photoinitiator (PI) were studied to optimize the conditions. It bypasses the first-pass effect of the liver, which reduces the dose before reaching the target.2 However, only a few drugs can be transdermal drug delivery candidates, because of their resistance to drug transport by the stratum corneum.3 This limitation could be overcome by micron-sized needles, which allow drugs to pass through the stratum corneum.Transdermal drug delivery has attracted immense attention in the pharmaceutical field, as it is a good alternative to the oral delivery system and traditional hypodermic injections.4 MNs can be made of metal, silicon, and polymers.5 Metal MNs, although mechanically strong and biocompatible, are dangerous because of the possibility of breakage underneath the skin. Microfabrication devices, such as micro-mechanical–electrical systems (MEMS), have used silicon as base material, suggesting its use for MN fabrication.6 Although the processing of silicon is well established, its complexity of fabrication could be a bottleneck for widespread use. Polymers, such as polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), and polyethylene glycol (PEG), which are biocompatible, could be used to fabricate MNs.7–9 Generally, a molding method is used for polymer fabrication.10 A mold is prepared using laser ablation, or microfabrication (such as photolithography), followed by dry etching. Subsequently, solidified polymers are obtained by filling polymers into the mold void. A mold with the shape of a hole array generates a polymer having the shape of an MN array. This method is useful for molding various kinds of polymers; however, it is difficult to fabricate complicated three-dimensional structures.11 Another fabrication method for polymers is the direct photopolymerization of prepolymers using a photomask.12–16 For monolithic photopolymerization, photocurable prepolymers mixed with photoinitiators (PI) are generally used. Prepolymers have ultraviolet (UV)-curable functional groups, such as acrylates, with double bonds that react with free radicals. After the UV exposure, PIs generate free radicals to polymerize prepolymers with acrylate groups.13 The degree of polymerization is controlled by varying the exposure time and PI concentration. A photomask induces selective photopolymerization with the transparent region of the mask transmitting a UV light. For instance, snake-fang-inspired MNs with multiple grooves can be realized by designing a photomask with multiple blade patterns.12 The groove can enhance the drug filling through MN surfaces owing to the capillary action. Another example is the fabrication of symmetric and asymmetric hollow MNs by one-shot photopolymerization with a designed photomask.14 By designing the photomask, MNs with particular shapes can be fabricated, which is an advantage of the direct photopolymerization.12–16 Many geometrical factors such as shape, tip diameter, height, force and velocity of insertion, and density of MN arrays and materials, should be considered before fabricating MNs.17 MN height is a particularly important geometric parameter that affects the possibility of stratum corneum penetration. Although there are several reports on the fabrication of MNs by photopolymerization, very few studies have investigated the effect of parameters, such as prepolymer molecular weight, and PI absorption, on the geometry of fabricated MNs. If the exposure time is too short, and the molecular weight of the prepolymer is low, the length of the MN is too short to penetrate the stratum corneum. If the exposure time is too long and the molecular weight of the prepolymer is too high, the polymerization region spreads in the horizontal direction, forming a film between the MNs, generating an effect that shortens the height of the MNs. In this study, conditions for fabricating high-aspect-ratio PEGDA were investigated. Polyethylene glycol diacrylate (PEGDA) is one of the widely used biocompatible hydrogel materials approved by the Food and Drug Administration (FDA).18,19 It can be used as a photocurable prepolymer for the fabrication of MNs by photopolymerization with a photomask.19 In this study, we investigated the condition for realizing high-aspect-ratio MNs by a photopolymerization to penetrate the stratum conium. Effects of the prepolymer molecular weight, and PI absorption coefficients on the fabrication of high-aspect-ratio MNs, were analyzed.Microneedles (MNs) enabling non-invasive and painless administration of drugs are one of the most promising transdermal drug delivery systems.Additionally, the design parameter, and the spacing ratio between MNs, to attain optimum conditions for MN penetration into the stratum corneum were studied. After MN fabrication on a flexible film, a penetration test was conducted on porcine skin.Hydrogel polyethylene glycol diacrylate  and 2-hydroxy-2-methylpropiophenone (Darocur 1173), 1-hydroxycyclohexyl phenyl ketone (Irgacure 184), and 2,2-dimethoxy-2-phenyl acetophenone (Irgacure 651) were purchased from Sigma-Aldrich, USA. PEGDA was mixed with 5 wt% of PI as a prepolymer. Polydimethylsiloxane (PDMS) was purchased from Dow Corning.A PDMS chamber was prepared and covered with a PET film. Then the PDMS chamber was filled with the prepolymer through the inlet hole. Next, a photomask was placed on the PET substrate and exposed to UV light. The uncured prepolymer was washed with ethanol.−2) was used to cure photocurable materials.SEM images were obtained using an EM-30 scanning electron microscope , with an acceleration voltage of 10 kV. A Fusion Cure 360  UV curing and patterning device . Absorbances were measured in the wavelength range of UV light (300–500 nm), and molar extinction coefficients were calculated using the Beer–Lambert equation.Cadaver porcine skin was obtained from a local butcher shop, cut, and secured to bare PDMS using pins. The coating solution was prepared using a mixture of sulforhodamine B and 1 wt% of sodium dodecyl sulfate (SDS) for uniform coating of MNs. The MNs were coated with the solution by 10 min immersion, dried for 2 h, and inserted into the skin for 10 min. The residual dye was removed after penetration.20 Then, in the propagation step, the unstable radicals stimulate the functional groups of the prepolymers and are added to the unsaturated chains.21 Subsequently, termination occurs in two ways: integration of two chains, which leads to the elimination of radicals (coupling), or the formation of two chains from two dead chains by hydrogen abstraction (disproportionation), stopping polymer chain elongation. In this study, PEGDA prepolymer with different molecular weights was synthesized as a biocompatible material and PIs, with different absorption coefficients, followed by prepolymer and PI mixing. Cone-shaped MNs were fabricated using photolithography, which is a simplified version of the conventional process using the principle of free radical polymerization (FRP). FRP is classified into three significant steps: initiation, propagation, and termination. In the initiation step, two radicals are generated from PI on exposure to UV light.22 Oxygen diffuses from the surroundings, hindering the reaction between radicals and prepolymers, by depriving them of radicals. This is the principle, by which cone-shaped MNs are fabricated through photopolymerization. The PDMS chamber, being an oxygen-permeable material, allows oxygen diffusion to the reaction sites, inducing a concentration gradient of oxygen. Additionally, an active inhibition reaction occurs at the reaction site, consuming oxygen, which contributes to the oxygen concentration gradient. Placing the reaction site between the oxygen-permeable (PDMS) and oxygen-impermeable (photomask) layers causes oxygen diffusion in both vertical and lateral directions. Thus, polymeric MNs form a cone shape from the central part of the oxygen-impermeable layer.23 UV light, refracted at the interface of the photocured and uncured PEGDA prepolymers, also leads to conical growth. As the cone angle becomes steeper, finally reaching a critical angle, the reflection remains only at the interface, and structural changes occur, as shown in 24Oxygen inhibition should be considered while using FRP to fabricate MNs.Xn) is calculated as follows:25ν is the kinetic chain length, which is the average number of monomer molecules added during polymerization, and k is the rate constant of termination . The molecular weight of the polymers was calculated as Mn = M0Xn. According to this equation, the high molecular weight of the monomer increases the final molecular weight. Thus, high prepolymer molecular weight indicates a higher possibility of fast solidification, because dissolution rates are related to molecular weights.26,27 We note that gourd-shaped MNs are observed in et al. reported the self-focusing effect in fabricating MNs by the reflection on the interface between the solidified and liquid prepolymer, and UV light rays diverged after reaching the critical point and converged again to form gourd-shaped MNs.24The average degree of polymerization  of each PI, the Beer–Lambert law was used:l is optical path length, and c is molar concentration.30 The molar extinction coefficient of Irgacure 651 was the highest, whereas the molar extinction coefficient of Darocur 1173 was the lowest. Thus, the MN growth rate during photopolymerization is proportional to the molar extinction coefficient of the PI used. PIs are very important for FRPs. A PI has a specific molar extinction coefficient value at a particular wavelength, which is one of the major factors affecting light absorption.In addition to studying the effect of chemicals on photopolymerization, geometric factors in the design of photomasks were investigated. An MN array with two different spacing ratios (diameter-to-spacing) was fabricated to examine growth rate and film formation. Two different photomask patterns, with the same diameter (150 μm), but different spacing ratios (diameter : spacing) of 1 : 1 and 1 : 2, were used. 31 In the current study, the UV exposure time was different for fabricating MNs with the same height. The short exposure time of PEGDA 700 for fabricating MNs without film formation induced a mechanical weakness because polymerization was not enough, although we conducted the additional UV exposure after rinsing to remove the uncured prepolymer.To study the mechanical stability of MNs with different PEGDA molecular weights, we conducted a penetration test with MNs of the same height. As shown in To investigate the penetration of high-aspect-ratio MNs, we examined the porcine skin after the penetration. A coating solution, prepared by mixing a dye (sulforhodamine B) and a surfactant (1 wt% of SDS), was used to confirm penetration. MNs were coated with the prepared solution using an immersion-coating method for 10 min and dried in air for 2 h. Fat and oil from the porcine skin were removed before penetration. MNs were inserted into porcine skin under 430 kPa for 10 min, and the residual dye was removed after penetration. In this study, conditions for the fabrication of high-aspect-ratio MNs, using photopolymerization with a photomask, were investigated. The effects of prepolymer molecular weight, PI absorption coefficients, and pattern spacing ratios on high-aspect-ratio MN fabrication were analyzed. High prepolymer molecular weights and PI molar extinction coefficients caused rapid height growth and film formation. Two different patterns with the same diameter (150 μm), and different spacing ratios (1 : 1 and 1 : 2), were used to study the geometric effects. Dense pattern arrays caused faster energy diffusion to the surroundings, owing to light scattering and diffraction. In the aspect of polymerization rate, PEGDA 700, patterns with a 1 : 1 spacing ratio, and Irgacure 651, are recommended for use. However, because penetrability is vital, PEGDA 700, with high fragility, is not suitable for use. A low molecular weight prepolymer (PEGDA 250) was used, for better robustness, even after the penetration test. High molar extinction coefficients induced a high MN growth rate, and short incubation time for film formation. Therefore, PEGDA 250 with high mechanical stability, and Darocur 1173 with high MN-formation controllability without film formation, and dense hole arrays, enabling higher growth rate, were optimum for penetration and hence MN fabrication. This work could guide prepolymer and PI selection, and enable tuning of the geometric factors of a photomask, to facilitate the use of new biocompatible materials to fabricate high-aspect-ratio MNs, and to design photomasks.There are no conflicts to declare.RA-012-D2RA00189F-s001


The aim of this study was to determine the horizontal burning rate, tensile strength, and surface morphology of kenaf fiber/PET yarn reinforced epoxy fire retardant composites. The composites were prepared by hybridized epoxy and Mg(OH)2 PET with different amounts of KF content  using the cold press method. The specimen with 35% KF (epoxy/PET/KF-35) displayed better flammability properties and had the lowest average burning rate of 14.55 mm/min, while epoxy/PET/KF-50 with 50% KF had the highest tensile strength of all the samples. This was due to fewer defects being detected on the surface morphology of epoxy/PET/KF-35 compared to the other samples, which influenced the mechanical properties of the composites.The application of natural fibers is rapidly growing in many sectors, such as construction, automobile, and furniture. Kenaf fiber (KF) is a natural fiber that is in demand owing to its eco-friendly and renewable nature. Nowadays, there are various new applications for kenaf, such as in absorbents and building materials. It also has commercial applications, such as in the automotive industry. Magnesium hydroxide (Mg(OH) In the last few decades, advanced application of composite materials has gained a lot of interest from various sectors, especially transmission towers, defense, aircrafts, marine, and the automotive industry ,2,3,4. INowadays, hybrid composites are widely used in industrial and automobile applications. A hybrid composite can be defined as a combination of two or more different types of fibers in which one fiber balances the deficiency of another fiber ,33,34,35Natural fibers contain hydrogen bonds and other linkages that give them strength and stiffness. In Western Europe, natural fibers are being used in the automotive industry and for packaging materials. Natural fibers are cellulosic materials, which have several fibrils that run all along the length of the fiber . The demOne of the potential constituents of natural fiber reinforced plastic composites in Malaysia is kenaf fiber (KF). Research on kenaf has grown tremendously. Kenaf long fibers could be used for a wide variety of applications if the properties were found to be comparable to existing synthesis composites. Nowadays, there are various new applications for kenaf, such as in furniture, composite boards, automotive panels, insulation mats, geotextiles, packaging, absorbents, building materials, and paper products. Kenaf also has commercial applications, such as in the automotive industry ,50,51. LWhen using natural fibers as reinforcement, the composite is prone to fire damage, and it is therefore essential to find additives that have low total emission of heat . TherefoCompared to other natural fibers, kenaf is eco-friendly and has a high growth rate. Therefore, in the present work, an investigation was carried out to study the horizontal burning rate, with magnesium hydroxide used to determine its capability as a fire retardant composite. Different percentages of kenaf fibers were added, with PET yarn and magnesium hydroxide used as controls. The objective was to determine the horizontal burning rate, tensile strength, and surface morphology of kenaf fiber/PET yarn reinforced epoxy fire retardant composites.The main materials used in the study were kenaf fiber and PET yarn. Kenaf fiber was ordered from Innovative Pultrusion Sdn. Bhd., Negeri Sembilan. First, the kenaf fibers were combed to clean and untangle the strong bonds between individual fibers. Combed fibers exhibit stronger mechanical properties compared to uncombed ones . Then, t3 was used as a binding material to fabricate the composite specimen. The recommended mixing ratio for epoxy resin and hardener is 2:1. The volume of epoxy used depended on the different ratios of kenaf fiber/epoxy, which determined the weight of epoxy needed. In this study, magnesium hydroxide was used as the control and a high filled-type inorganic flame retardant, with 5% magnesium hydroxide mixed with the epoxy resin to enhance the flame retardancy of the composite material.The composite comprised a hybrid of kenaf fiber/PET yarn as reinforcement, while epoxy resin was used as the matrix and magnesium hydroxide as the fire retardant . Epoxy wThe composite samples were prepared using a hybrid of kenaf fibers/PET yarn with the epoxy resin as binder and magnesium hydroxide as fire retardant through a random hand lay-up technique. The composition of the samples of kenaf fiber reinforced epoxy fire retardant composites is shown in Before fabricating, kenaf fibers were combed and then cut into 22 cm pieces. After that, the fibers were weighed based on the density of the fiber to determine the exact portion of fiber that should be used in the fiber polymer composite samples to obtain the designated fiber volume content. One of the problems with kenaf fiber reinforced epoxy that needs to be addressed is uneven fiber distribution . In addiThe fabricating process of the composites in this study was as follows. The molds were polished using grease oil (releasing agent) before starting fabrication. Epoxy resin was mixed with a ratio of 2:1 (resin: hardener by weight) according to the manufacturer’s specifications. The mixture was stirred slowly for 10 minutes to ensure that both epoxy and hardener were uniformly mixed. Then, magnesium hydroxide was poured into the mixture of epoxy and hardener. The mixture of magnesium hydroxide was then stirred slowly for 5–10 min to ensure the mixtures were uniformly mixed.A transparent plastic sheet was placed on the surface of the mold. Then, the mixture of magnesium hydroxide with epoxy and hardener was poured slowly into the mold. Next, the fibers and PET yarn were completely immersed in resin by portion and then distributed randomly in the mold. The composite was covered by a transparent plastic sheet to avoid formation of air bubbles inside the mold. The plastic cover was used to prevent the fibers from attaching to the steel plate during curing. The composite was pressed using a flat plate to remove the remaining bubbles inside the mold. The mold was then compressed by another steel plate on top of the transparent plastic sheet. Demoulding of specimens was carried out after 24 h of fabrication. For each composition, the fiber and its binder were calculated to determine the weight of the fiber and epoxy needed to fill the mold. After fabrication of the composite was complete, the specimen of each composition was cut into nine pieces of 1.3 cm width for use in the horizontal burning test and eight pieces of 1.5 cm width for use in the tensile test. The images of these composites were also captured after the tensile test. The burning test was conducted to determine the burning rate of the samples. The samples were set up according to ASTM standard D635. For these points, all specimens were cut to exact length with dimension of 12.5 cm length × 1.3 cm width × 0.3 cm thickness. The test was carried out in triplicate. The test samples were supported horizontally at one end. Next, the free end was exposed to a specified gas flame for 30 s. The time and extent of burning were measured using a stopwatch, and it was noted if the specimen did not burn. An average burning rate was noted for the material if it was burned to the 100 mm mark from the ignited end. The formula below was used to determine the average burning rate of each sample:The fiber tensile test was used to determine the strength of the composite materials. The samples were set up according to ASTM standard D3039M. First, all of the samples were cut into the same size of 20 cm height × 1.5 cm width × 0.3 cm thickness. Then, four aluminum plates with a dimension of 25 mm × 15 mm × 1 mm were attached to the two sides of both ends of the samples using the epoxy resin. The test was carried out in five replications using the Universal Testing Machine (INSTRON 5556)  with a 5 kN load cell; the crosshead speed was maintained at 2 mm/min.SEM is capable of performing analyses of selected point locations on a sample, which is especially useful for qualitatively or semiquantitatively determining chemical composition (using energy-dispersive (EDS)), crystalline structure, and crystal orientations (using electron backscatter diffraction (EBSD)). The design and function of the SEM are very similar to EPMA, and considerable overlap in capabilities exists between the two instruments. From the tensile test fracture samples, the samples at the edge of the fracture side were cut using a jigsaw machine. Then, the samples were coated with gold using a vacuum sputter coater to make them conductive prior to SEM observation. The cross section of samples was coated with gold as shown in From the results of horizontal burning test for every sample, the average horizontal burning rate was determined. From The KF content for epoxy/PET/KF-20, epoxy/PET/KF-35, and epoxy/PET/KF-50 was 20%, 35%, and 50%, respectively. The average burning rate of epoxy/PET/KF-35 was the lowest at 14.553 mm/min compared to the other samples. As a result, the composite with 35% KF and 5% magnesium hydroxide showed better result due to the compatible combination of fiber and fire retardant compared to 20% and 50% KF. The fiber volume content in epoxy/PET/KF-35 was the best compared to epoxy/PET/KF-20 and epoxy/PET/KF-50 samples because of the lower burning rate and higher fire retardant effectiveness. When the fiber content was increased above 20%, the characteristics of the composite became more similar to lignocellulosic materials . A recenThe flame took a long time to propagate along epoxy/PET/KF-35 and also produced char at the same time. The production of char was the reaction of Mg in front of the flame. Charring is known as a chemical process of incomplete combustion of certain solids when subjected to high heat. By the action of heat, charring removes hydrogen and oxygen, so only carbon remains in the char. In addition, as acid and halogen-free flame retardant, Mg releases the water of hydration when it decomposes endothermically, which helps the flame retarding action . The eneThe ultimate tensile strength, elongation break, and elastic modulus were determined from the stress–strain curve of hybrid kenaf fiber/PET yarn reinforced epoxy fire retardant composite after the test. Statistical analysis was conducted based on the data of five specimens for each composite sample. The tensile strength of hybrid kenaf fiber/PET yarn increased with the increase of fiber volume content, as shown in According to the study by Mobedi et al. , the addThe surface morphology of all the specimens after the tensile test was studied by capturing images of the fractured area. From this analysis, it was concluded that several types of defects occurred on the specimens. Defects typically take place during the production of composites and sometimes by manual construction known as “lay-up”. Variations in the amount of defects in composites during manufacturing will increase the likelihood of composite failure.2 has a great tendency to form agglomerates. In fact, agglomeration is a well-known phenomenon, and its probability increases with decreasing particle size. These agglomerated particles could be stress concentrator points and could affect the final performance of the composites. This might result from the defects that occur in the composites during fabrication. Moreover, it is possible to identify the needled structure of Mg(OH)2 particles in images of higher magnification, e.g., 950×. This behavior might be an indication of poor interaction between the matrix and Mg(OH)2 [Two problems are critical to the material response: defects (built-in at manufacture) and damage (changes due to use). The effects of damage have been extensively covered in the open literature. Heslehurst and Scott  found th Mg(OH)2 .As shown in In order to reduce cost and provide a pollution-free environment, the flammability and mechanical properties of hybrid kenaf fiber/PET yarn reinforced epoxy fire retardant composites was analyzed. The horizontal burning test showed that epoxy/Mg/PET with 0% KF had the highest average burning rate of 22.151 mm/min compared to epoxy/PET/KF-20, epoxy/PET/KF-35, and epoxy/PET/KF-50, which contained 20, 35, and 50% KF, respectively. The average burning rate of epoxy/PET/KF-35 was the lowest at 14.553 mm/min compared to the other samples. This suggests that epoxy/PET/KF-35 would be better for application as a fire retardant composite because of the optimum composition. Epoxy/PET/KF-50 demonstrated the highest tensile strength of 32.022 MPa compared to the other samples. The tensile strengths of epoxy/Mg/PET, epoxy/PET/KF-20, and epoxy/PET/KF-35 were 10.867, 19.956, and 25.244 MPa, respectively. The tensile strength of the hybrid kenaf fiber/PET yarn increased with the increase of fiber volume content. The combination of kenaf fiber and PET yarn enhanced the tensile properties of the composites. In addition, too many defects result in poor mechanical properties of composites. Epoxy/PET/KF-50 showed the fewest defects. In general, the surface morphology of the hybrid kenaf fiber/PET yarn possessed defects such as fiber pull-out and debonding.


In this review, we discuss prevailing models of NMDs and highlight the current progress and ongoing challenges in developing human iPSC-derived (hiPSC) 3D cell culture models of functional NMJs. We first review in vivo development of motor neurons, skeletal muscle, Schwann cells, and the NMJ alongside current methods for directing the differentiation of relevant cell types from hiPSCs. We further compare the efficacy of modeling NMDs in animals and human cell culture systems in the context of five NMDs: amyotrophic lateral sclerosis, myasthenia gravis, Duchenne muscular dystrophy, myotonic dystrophy, and Pompe disease. Finally, we discuss further work necessary for hiPSC-derived NMJ models to function as effective personalized NMD platforms.The neuromuscular junction (NMJ) is a specialized cholinergic synaptic interface between a motor neuron and a skeletal muscle fiber that translates presynaptic electrical impulses into motor function. NMJ formation and maintenance require tightly regulated signaling and cellular communication among motor neurons, myogenic cells, and Schwann cells. Neuromuscular diseases (NMDs) can result in loss of NMJ function and motor input leading to paralysis or even death. Although small animal models have been instrumental in advancing our understanding of the NMJ structure and function, the complexities of studying this multi-tissue system  The NMDs primarily involve dysfunction of motor neurons (MNs), skeletal muscle (SkM), or their synaptic connection, the neuromuscular junction (NMJ). Different NMDs have distinct tissue origins such as MNs , muscle , NMJs , or a combination thereof . Regardless of the origin, the structural and/or functional deficit in a targeted tissue will resonate throughout the entire motor unit, leading to multiple shared symptoms among different NMDs. NMDs are estimated to affect 160 per 100,000 people worldwide ; howeverapeutics . Animal in vivo, limited capability to isolate neural stem cells, and the postmitotic nature of adult MNs complicating in vitro studies. Over the past decade, advances in human induced pluripotent stem cell (hiPSC) technology, have provided a novel source of human somatic cells for pre-clinical research. hiPSCs can be generated from ethical and accessible sources, such as the skin and blood, and function as a potentially unlimited, patient-specific source of traditionally inaccessible cells such as MNs and cardiomyocytes. Additionally, hiPSC-derived cells can be integrated into two- (2D) and three-dimensional (3D) culture systems to enable novel studies of human development, disease, and pharmacology. hiPSC-based derivations of MNs and SkM, in particular, provide easily accessible, highly expandable sources of patient-specific NMD-relevant tissues. Notably, these platforms complement animal models creating an efficient and predictive system for patient specific NMD modeling and drug development. Incorporation of hiPSC-derived MNs and SkM into 3D cultures and organ-on-a-chip systems adds necessary structural complexity and genetic and environmental control over the cell-specific behavior. These tissue-engineered motor units recapitulate the nature of functional NMJs offering potential for improved mechanistic understanding of complex NMDs  development and draw parallels to current methods to differentiate these cells from hiPSCs. We finalize this section by describing NMJ development and function.Over the past decade, methods to differentiate hiPSCs into neuronal cells has rapidly progressed due to increased understanding of early neural development and commitment of neuroprogenitor cells to highly specialized neural subtypes, including MNs . MNs areCells of the developing vertebrate nervous system are derived from the ectoderm which forms during gastrulation . InhibitRegionalization is first specified in the brain with cells assuming a rostral forebrain identity in the absence of morphogens. These cells continue to develop into the neurons comprising the telencephalic region of the brain, which houses the cerebral cortex where mature UMNs reside . The remNeural precursors are also subject to dorsal–ventral patterning concurrent to rostral–caudal patterning. WNTs and BMPs derived from roof plate cells mediate dorsal patterning . In contLower motor neurons at this stage are referred to as having a general character until organized into distinct motor columns that correspond to targeted regions of innervation. These regions include the median motor column (MMC), which innervates axial SkM, spinal accessory column (SAC), which innervates the branchial SkM of the face and neck, phrenic motor column (PMC), which innervates the SkM of the diaphragm, lateral motor column (LMC), which innervates appendage muscle, hypaxial motor column (HMC), which innervates intercostal and abdominal SkM, and preganglionic motor column (PGC), which synapse onto ganglionic neurons of the autonomic nervous system (ANS) . Each moOver the past decade, a variety of protocols have been used to derive MNs from hiPSCs. These methods vary in length and efficiency; however, they are designed on the genetic and molecular principles of embryonic development . HistoriAfter neuralization, neural precursors are committed to MN progenitors by following common patterning principles of caudalization and ventralization. MN differentiation efficiency and culture length have been improved by optimizing the concentrations and timing of patterning morphogens like WNTs, BMPs, RA, FGFs, and SHH . Althougin vivo  begins with the paraxial mesoderm . The parin vivo . In the in vivo  and othein vivo . Cells win vivo . Differein vivo .T and expression of Msgn1 and Tbx6  fate characterized by the downregulation of and Tbx6 . In the and Tbx6 . As skeland Tbx6 . At the egulated . Within egulated . Further somites . Shortlypression . Soon afon , and galactosylceramidase  are unable to form NMJs; however, these mice can form aneural AChR clusters on muscle fibers prior to innervation  that shares homology with the WNT receptor, Frizzled. As a result, WNT proteins bind and activate MuSK prior to innervation, when neural agrin is absent . This siAfter forming functional NMJs with their target, MNs impinge on muscle fiber structural and functional diversity. A single muscle is composed of several fiber types that are innervated by specific classes of MNs. MNs are subdivided into three groups based on the type of muscle fiber they innervate: (1) alpha MNs, which innervate force generating extrafusal fibers, (2) gamma MNs, which innervate the proprioceptive intrafusal fibers, and (3) beta MNs, which innervate both extrafusal and intrafusal fibers. Alpha MNs are the most abundant of these classes and are categorized as SFR , FFR , and FF  reflecting the type of extrafusal muscle fiber they innervate . MNs areCaenorhabditis elegans  or polycaprolactone (PCL) nanofibers, served to align murine myoblasts and improve NMJ formation with co-cultured rat embryonic spinal MNs . Micropain vitro while supporting longer, myelin ensheathed axonal projections in rodent models in vivo , optogenetic control  to rescue MN function through overexpression within the SkM  as a potential ALS therapeutic agent  hiPSC-derived neural spheroids within a microfluidic device  which shortens telomere length and induces a more severe SkM  and II (DM2). DM1 results from a series of CTG repeats in the DM protein kinase (DMPK) gene, while DM2 results from a series of CCTG repeats in the Zinc Finger 9 (ZNF9) gene . The majAberrant splicing is not limited to SkM but occurs in multiple organs including the cardiac and neurological systems . In DM1,LR model), but this does not lead to muscle wasting or denervation  perturbations , is a rare metabolic autosomal recessive disorder that results from deficiency of acid α-glucosidase (GAA) . Pompe dIn Pompe patients and mice, glycogen accumulation occurs in the brain , centralPompe disease has been predominantly modeled in the GAA knockout mice  that recCurrent clinical therapy for Pompe disease is enzyme replacement therapy (ERT) which systemically delivers the recombinant human GAA (rhGAA) to break-down accumulated glycogen. While ERT significantly prolongs patient lifespan and augments quality of life , it is lin vitro human cell culture systems play important roles in modeling genetic diversity and neuromuscular dysfunction in Pompe disease. Specifically, human myotube cultures accurately model clinical differences between IOPD and LOPD patients , and endothelial cells will be required to recreate native NMJ complexity. Here, heterocellular interactions can stimulate formation and advanced maturation of biomimetic NMJs to model both normal physiology and disease. For example, SCs are essential in the formation and maintenance of the adult NMJ  and theiin vivo  as well in vitro . Their rin vitro NMD models and platforms for predictive drug screening will also require the incorporation of cell of the immune system and cells involved in drug metabolism, respectively. The immune system is a critical regulator of both neurological  modules containing extramuscular tissues such as intestine, gut, liver, and fat. These multiplexed culture systems will be able to identify unexpected drug toxicities due to organ-organ crosstalk, such as that seen for bleomycin cardiomyocyte toxicity only in the presence of lung tissue (Generation of advanced ological  and muscological  homeostaological , T lymphological , and masological  at NMJs.ological . Realistg tissue . Multiplg tissue . While ain vivo 3D microenvironments of developing or diseased NMJs. For example, 3D hydrogels that incorporate the basement membrane proteins laminin and collagen IV have been shown to support increased contractile force generation in muscle-only (Beyond incorporation of additional cell types, further advancements in biomaterial design and microfabrication technologies will be critical for successful engineering of microphysiological systems that replicate cle-only  and musccle-only  culture cle-only . Recent cle-only , combinecle-only , offer ocle-only  of bioaccle-only , along wcle-only , could pin vitro NMJ models are mainly utilized for small-scale, stand-alone studies, or to supplement whole-organ and organism-level in vivo investigations with human cell- and tissue-specific data. Combining animal studies from a single genetic background with in vitro validations in hiPSC-based models from genetically diverse patients are likely to improve the predictive value of preclinical therapeutic tests. Eventually, however, advances in hiPSC technology along with miniaturization and standardization of microphysiological systems are expected to enable self-sufficient, high-throughput in vitro pharmacological screens with directly translational outcomes. Altogether, we anticipate that future advances in patient-specific hiPSC-based in vitro modeling of NMDs will be instrumental for gaining deeper understanding of human NMD pathophysiology and will lead to streamlined developments of pharmacotherapies for these devastating disorders.Current ZF, EL, and TC wrote the manuscript. TC and ZF generated figures for the manuscript. AK and NB edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


GOx is presently known to be produced only by fungi and insects. The current main industrial producers of glucose oxidase are Aspergillus and Penicillium. An important property of GOx is its antimicrobial effect against various pathogens and its use in many industrial and medical areas. The aim of this review is to summarize the structure, function, production strains and biophysical and biochemical properties of GOx in light of its various industrial, biotechnological and medical applications.Glucose oxidase (GOx) is an important oxidoreductase enzyme with many important roles in biological processes. It is considered an “ideal enzyme” and is often called an oxidase “Ferrari” because of its fast mechanism of action, high stability and specificity. Glucose oxidase catalyzes the oxidation of  Glucose oxidase is an enzyme that has widespread applications in industry and biotechnology. Due to this, a deep understanding of its structure and function are warranted. Glucose degradation is the most universal metabolic process. In addition to its breakdown in glycolysis, glucose can also be directly oxidized to glucono-d-glucose: NAD(P)+ 1-oxidoreductase, E.C. 1.1.1.47) [d-glucose:ubiquinone oxidoreductase, E.C. 1.1.5.2) [d-glucose:oxygen 1-oxidoreductase, E.C. 1.1.3.4) [These fall into two classes: 1) the dehydrogenases glucose dehydrogenase  (NAD(P)orm H2O2 . The priThe high specificity, high turnover and high stability of GOx make it an ideal enzyme for biosensor applications , some ofGOx is a member of the glucose-methanol-choline oxidoreductase (GMC oxidoreductase) superfamily. The members of this family are all FAD-dependent oxidoreductases that share a common fold ,8. They A. niger and one from P. amagasakiense [A. niger GOx structures range in resolution from 2.4–1.2 Å , potentially allowing those sites to be identified in the crystal structure [More particularly, a study using H/D isotope substitution found that reduced glycosylation appeared to lower the enthalpy of activation and, therefore, increase the activity of the enzyme . Deglycotructure .A. niger GOx ; seven of these sites have been confirmed in at least one of the known A. niger GOx structures. Asn-43 does not appear to be glycosylated in any of these structures, and peptide sequencing work also suggested that no carbohydrate is attached here [Potential glycosylation sites can be identified by the consensus sequence N-X-T/S, where X is any amino-acid residue except proline . Eight shed here .Burkholderia pseudomallei [Acetobacter suboxydans [GOx is primarily produced by fungi and insects . There wdomallei  and Acetboxydans  may be dAll these studies made use of what would now be considered rather crude cell-free preparations, which would make it difficult to distinguish between the glucose oxidase activity of GOx and that of pyranose oxidase. To date, there appears to have been no confirmed glucose 1-oxidase identified in any bacterial strain, and a recent phylogenomic study of the GMC oxidases found that pyranose oxidase is only one of the GMC proteins present in both fungi and bacteria .Streptomyces coelicolor alditol oxidase, E.C. 1.1.3.41, was incorrectly classified as GOx in one of these databases), a total of 50 unique species were identified; 45 species were from fungi, and five were from insects.To unambiguously identify those organisms that are presently known or suspected of harboring GOx, the NCBI Protein database, UniProtKB and the Brenda Enzyme Database were searched for entries classified as having E.C. number 1.1.3.4, and some of the recent literature was surveyed. After discarding duplicate entries and removing those that were mis-annotated  [Helicoverpa armigera (Cotton bollworm) [Heliothis viriplaca , Mythimna separata  and Spodoptera exigua (Beet armyworm) [The insect species include ney bee) , Helicovollworm) , Heliothrmyworm) . No mammA. niger GOx according to BLAST. All sequences are roughly the same length  and the alignment covers at least 89% of the A. niger sequence. They can be divided into three main groups: The two Aspergillus species share 85% identity, next are the Penicillium and Talaromyces species with 63–67% identity to A. niger (with one exception), and last are all other species, which have only 27–35% identity.Only 27 of the 50 species had sequences available in one of these three databases. A. niger GOx and other, functionally non-equivalent GMC oxidases. This means that it will generally not be possible to identify as GOx uncharacterized sequences from more distantly related organisms even within the same kingdom using BLAST searches alone.As shown in Aspergillus, Talaromyces, and most of the Penicillium species, while the other branch contains all the remaining species. Curiously, the Yarrowia sp., Thanatephorus cucumeris, and Ceratocystis fimbriata species are closer to the insect species than the fungal species.A phylogenetic tree based on an alignment of all these sequences generally maintains the grouping seen in A. niger, P. amagasakiense and M. restricta) and two insect (A. melifera and S. exigua) GOx sequences highlights the conserved areas  of the The A. melifera. These substitutions suggest that there are subtle differences in the conformation of the active sites in these proteins; they also provide additional support to the designation of His-516 as the primary catalytic residue as described in In the insect sequences, His-559 is replaced with an asparagine , while Glu-412 is replaced with an aspartate in A. niger and P. amagasakiense structures, are conserved only in these two sequences. The cysteines in M. restricta, A. melifera and S. exigua appear in different locations in the sequence, suggesting either differences in disulfide bond formation or no disulfide bonds occur in these proteins.The sequence alignment also shows that the positions of Cys-164 and Cys-206, which form disulfide bonds in the A. niger and P. amagasakiense glycosylation sites identified structurally  glycosylation motifs in the neighborhood of residues Asn-355 and Asn-388, suggesting that at least one glycosylation site might be expected in this location.Finally, it may be worth noting that the GOx is used in many branches of industry because of its ability to oxidize glucose and produce hydrogen peroxide. Its rapid turnover and high stability finds it many applications in the food, pharmaceutical, medical, textile and power industries. For many of these applications GOx is used in a biosensor or nanosensor ,97,98,99In many modern applications, GOx is often used in combination with other enzymes, for example, tyrosinase in the analysis and discrimination of musts and wines , α-amyland bread , peroxidnd bread  and tearnd bread , the autnd bread  and insund bread ,108. Finnd bread .The applicability of GOx primarily depends on its quantity, thermal stability and activity. Many studies focused on identifying which fungal strains are better for biosensor development, which are better for clinical studies and which are better for biochemical diagnostic tests . OptimalAspergillus and Penicillium [A. niger and P. amagasakiense. These genera are important because of their metabolic versatility and because they are “generally regarded as safe” by regulatory agencies [Industrially, the most important producers of GOx are species belonging to the genera icillium ,110,111,agencies .P. amagasakiense GOx is catalytically more effective than A. niger GOx, with a d-glucose and A. niger and P. amagasakiense GOxes [A. niger GOx.Overall, kcat/Km) ; howeverkcat/Km) . This prse GOxes . Other rse GOxes  and therse GOxes  of A. niAspergillus tubingensis CTM507 produced by SSF and SmF found that SmF produced GOx more efficiently but that the GOx produced by SSF had a higher activity  and submerged fermentation (SmF) ,119. Subfor SmF) . Both offor SmF) .A. niger by glucose [A. niger GOx include the Agp-chloro-mercapto-benzoate, phenyl mercuric acetate [Glucose and saccharose are the carbon sources most often used for the industrial production of GOx ,121. GOxucose [3 , Mn2+, Cnic acid , EDTA , which gradually releases a controlled amount of hydrogen peroxide ,184. HowFurthermore, the high concentration of HThe development of nanozymes, such as metal-based nanoparticles and carbon-metal hybrid nanomaterials, which can simultaneously exhibit dual or multienzyme mimetic activity, are a promising antibacterial therapy in wound care.Nanozymes with embedded GOx have very recently become the object of several studies investigating their wound-healing properties ,186,187.Staphylococcus aureus compared to unjoined FeInterestingly, in neutral and acidic microenvironments, it exhibited pH-switchable GOx/peroxidase and GOx/catalase cascade reactions, respectively. Thus, a fabricated FeAlthough these nanozymes show effective antibacterial and wound healing properties, their direct use in clinical settings is considered questionable. Their unique characteristics, including their small size, chemical composition and solubility, may create a high risk and a hazard for human health. To address the limits of nanozymes in wound care, nanozymes can be incorporated into suitable wound dressings .Zhang and co-workers  developed-glucose to d-glucono-d-gluconic acid and hydrogen peroxide in the presence of molecular oxygen. Due to its fast turnover and high stability and specificity, glucose oxidase has found many uses in industry and medicine.Glucose oxidase catalyzes the oxidation of Uses have been found for all aspects of its reaction: its consumption of glucose, production of HA. niger GOx. Although a number of industrial strains with enhanced stability or activity have been prepared, there is no structural analysis of what makes these forms more stable. Despite numerous efforts, there is still no structure of a GOx–substrate complex.Although the biochemical and biophysical characteristics of GOx have been thoroughly studied, there are still areas where additional study would be beneficial. For example, all but one of the structures of GOx presently known are variations of Moreover, some uncertainty still remains as to whether GOx might also be found in some bacteria or whether animals other than insects might produce it. Industrial research is also expected to develop new materials for biosensors and nanosensors and new ways of attaching GOx to them. In addition, GOx represents a promising molecule, which, in the form of nanozymes incorporated into wound dressings, may be of great use in wound care.


The previous clinical treatment for CRC mainly involves conventional surgery, chemotherapy, and radiotherapy. With the development of tumor molecular targeted therapy, small molecule inhibitors present a great advantage in improving the survival of patients with advanced CRC. However, various side effects and drug resistance induced by chemotherapy are still the major obstacles to improve the clinical benefit. Thus, it is crucial to find new and alternative drugs for CRC treatment. Traditional Chinese medicines (TCMs) have been proved to have low toxicity and multi-target characteristics. In the last few decades, an increasing number of studies have demonstrated that TCMs exhibit strong anticancer effects in both experimental and clinical models and may serve as alternative chemotherapy agents for CRC treatment. Notably, Wnt/β-catenin signaling pathway plays a vital role in the initiation and progression of CRC by modulating the stability of β-catenin in the cytoplasm. Targeting Wnt/β-catenin pathway is a novel direction for developing therapies for CRC. In this review, we outlined the anti-tumor effects of small molecular inhibitors on CRC through Wnt/β-catenin pathway. More importantly, we focused on the potential role of TCMs against tumors by targeting Wnt/β-catenin signaling at different stages of CRC, including precancerous lesions, early stage of CRC and advanced CRC. Furthermore, we also discussed perspectives to develop potential new drugs from TCMs  These findings establish a direct link between Wnt/β-catenin signaling pathway and human CRC. Furthermore, more than 90% of sporadic CRCs has been identified to carry mutations of one or more components of the Wnt/β-catenin signaling pathway including APC based on the genome-scale analysis  is the third cause of cancer-related death worldwide according to the latest statistics of the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) . It esti in 1991 . Then soanalysis . TherefoIn clinical practice, standard conventional treatments for CRC are surgery, chemo-therapy and radiotherapy. Currently, with the development of tumor molecular targeted therapy, small molecule inhibitors present a great advantage in improving the survival of patients with advanced CRC. Moreover, long-term application of these therapies can lead to various side effects and toxicities, consisting of nausea, vomiting, mucositis, peripheral neuropathy, and diarrhea . Thus, iβ-catenin pathway is mainly related to the stability of ß-catenin controlled by the ß-catenin destruction complex that is comprised of scaffolding proteins APC, Axin and the kinases casein kinase 1 (CK1) and Glycose synthase kinase 3β (GSK3β). Absence of Wnt ligands stimuli, the cytosolic ß-catenin is phosphorylated by GSK3β, ubiquitinated by ß-TrCP200 and targeted for proteasomal degradation. The ligand Wnt binds to the cell surface receptor Frizzled and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) to form a trimer, which recruits the Dishevelled (Dvl) protein to the plasma membrane, leading to dissociation of the destruction complex followed by cytosolic accumulation of ß-catenin. Consequently, the ß-catenin translocates to the nucleus where nuclear ß-catenin cooperates with TCF/LEF family transcription factors to active target genes such as c-myc, MMP-7, SNAIL and EGFR  and non-canonical (β-catenin independent) pathways . Originaand EGFR . The actand EGFR .in vivo  is a crucial process by which epithelial cells lose cell polarity and cell-cell adhesion, and closely associate with invasion and metastasis in many types of malignancies including CRC . There iinvasion .+ stem cells can increase additionally the population of Lgr5-positive cells and drive adenoma expanding in colon  theory underlies tumor proliferation, differentiation and metastasis. Although there is still no consensus on the concept of cancer stemness, the vital role of the Wnt pathway for the function of normal and cancer stem cells is commonly accepted . In the in colon . CD44v6,in colon . Moreovein colon . Recentlin colon . Taken tß-catenin is a critical event in CRC progression. Porcupine (PORCN) is a membrane-bound O-acyltransferase protein which regulates Wnt ligands secretion outside the cell membrane through palmitoylation. In recent years, PORCN has emerged as a molecular target for treating Wnt-driven cancers. ETC-159, WNT974 (LGK974) and Rxc004 has been identified as potent inhibitors of Wnt secretion inhibiting ß-catenin activity in preclinical studies. ETC-159 has been proven to be remarkably efficacious in treating CRCs with R-spondin translocation in vivo and in vitro experiments  domains of TNKS in DLD-1 and SW480 cell lines in vitro (Some studies indicate that tankyrases (TNKS) are novel targets for Wnt inhibition by regulating stabilization of Axin and hence leading to increased in vitro . JW55 alin vitro . G007-LKin vitro . In partin vitro . IWR-1 iin vitro .ß-catenin levels. Ewan K et al. revealed that three small molecule inhibitors including CCT031374, CCT036477, and CCT070535 can block the Wnt/β-catenin signaling through reducing the level of ß-catenin without altering its stability, which is different from drugs involving inhibition of TCF-dependent transcription in SW480 cells , is a precursor and a major risk factor for CRC . Currentwith FAP . In addiwith FAP .ß-catenin, leading to inactivation of the Wnt/β-catenin pathway in a CRC xenografted mouse model and HT-29 cell line. Brucine and strychnine from nux vomic have remarkable effects in improving circulatory system and relieving arthritic and traumatic pains. Recently, ß-catenin. Meanwhile, they can greatly promote DKK1 expression, which is proved to negatively regulate Wnt/β-catenin pathway. On the other hand, some monomers derived from traditional Chinese herbs such as Luteolin, C. brachycephalum, pterisolic acid G (PAG), wogonin, nerigoside (NG) and isobavachalcone (IBC), exhibit anticancer functions by affecting the phosphorylation state of GSK-3β and ß-catenin in CRC. However, nerigoside has been found to destroy the balance of proliferation and apoptosis through the ERK/GSK3β/β-catenin signaling pathways, whereas isobavachalcone exerts its anticancer effect via the AKT/GSK-3β/β-catenin pathway in CRC , 1,4,6-Tri-O-galloyl-β-d-glucopyranose (TGG) as well as tetrandrine (TET) could suppress colorectal tumor growth and downregulate target genes expression  anthocyanins can reduce the expression of miR-483–3p accompanied by an increased level of DKK3 expression, which is one negative regulator of Wnt pathway  could decrease nuclear lcatenin and downregulate the transcriptional activity of ß-catenin/Tcf, leading to inhibition of cell proliferation in SW480 cell lines  . IPM711,As well as the monomers and extracts derived from TCMs, an increasing body of evidence suggests that TCM formulas possess anticancer properties, too. Zuo Jin Wan (ZJW) has been used in the treatment of gastrointestinal and liver diseases in China for ages , which iin vivo, but not in vitro experiments  through canonical Wnt signaling. It is showed that apple polysaccharide (AP) from apple residues could affect the activation of Wnt/β-catenin signaling pathway eriments . PreviouCRC is often diagnosed at an advanced stage when tumor cell dissemination has already taken place and chemotherapy was one of the major methods for the treatment of CRC in the past few decades. In clinic, it is obviously clear that fluoropyrimidines, irinotecan, and oxaliplatin have been widely applied to chemotherapeutic regimens for tumors . The recin vitro studies suggest that resveratrol exhibited preventative colon cancer effects and this was associated with Wnt signaling (93). In this clinical trial, patients with colon cancer were randomly provided a treatment with resveratrol, and relevant studies tested its effects directly on colon cancer and normal colonic mucosa. These results showed that resveratrol could inhibit Wnt/β-catenin signaling in the normal colonic mucosa, but not in colon cancer (On account of the significance of Wnt/β-catenin pathway in CRC development and metastasis, some native components of TCMs was developed as novel drugs specifically targeting this signaling pathway and are already in clinical trials . Resveran cancer . Thus, rn cancer .-5a and thereby play the anti-metastatic role. The safety and tolerability of Foxy-5 were established and early signs of anti-metastatic activity were evaluated in subjects with resectable colon cancer. Further, researchers have already examined the maximum tolerated dose and dose-limiting toxicity of this drug (Besides, small molecular weight Wnt 974, a potential inhibitor of Wnt/β-catenin signaling, has been used to assess its safety and antitumor activity in combination with chemotherapeutic agents in patients with BRAF-mutant metastatic CRC and Wnt pathway mutations . Neverthhis drug . Intereshis drug .ß-catenin/TCF4 pathways which represent the main elements of the Wnt/β-catenin pathway involved in the treatment of CRC. Thus, understanding the molecular mechanisms of action of TCMs and how they target Wnt/β-catenin may shed light on future therapies for CRC. However, it needs multi-level and multi-link comprehensive action to anti-tumor because of the complex composition of traditional Chinese medicine. This suggests that we need to investigate the crosstalk between Wnt/β-catenin signal pathway and others. In addition, there remains very few new clinical treatments under development due to lack of strict evaluation system for effectiveness and safety of TCMs. Therefore, it will hopefully pave the way for the CRC clinical treatment and may also relieve the side effects related to chemotherapy if there is a breakthrough in the study of multi-target intervention of TCM in CRC.CRC has become a global public health problem on account of its high incidence and mortality rate worldwide. The clinical treatments for CRC mainly involve surgery-based chemotherapy. In recent years, with the application of targeting small molecules against cancer, the quality of life for CRC patients has improved. Nevertheless, chemotherapy-induced side effects and drug resistance remain a major issue for clinical practice. Numerous studies have shown that TCMs can be used to exert potential anticancer activity and alleviate the side effects associated with chemotherapy. It is confirmed that various mutations in one or more members of the canonical Wnt signaling pathway take place in the progression of CRC. Therefore, in this review, we aimed to intensively explore molecular mechanisms of TCMs against cancer at the different stages in CRC progress, including precancerous lesions, early stage CRC and CRC invasion and migration based on the inhibition of the Wnt/β-catenin signaling pathway. Cell culture and animal experiments have found that TCMs play anticancer roles by regulating APC/β-catenin, GSK-3β/β-catenin, and


It was found that the expression of N-cadherin was reversely related to the expression of odontogenic markers  during the differentiation process of DPSCs. Specific shRNA-mediated knockdown of N-cadherin expression in DPSCs significantly increased the expression of DSPP and Runx2, alkaline phosphatase (ALP) activity, and the formation of mineralized nodules. Notably, N-cadherin silencing promoted nucleus translocation and accumulation of β-catenin. Inhibition of β-catenin by a specific inhibitor XAV939, reversed the facilitating effects of N-cadherin downregulation on odontogenic differentiation of DPSCs. In addition, knockdown of N-cadherin promoted the formation of odontoblast-like cells and collagenous matrix in β-tricalcium phosphate/DPSCs composites transplanted into mice. In conclusion, N-cadherin acted as a negative regulator via regulating β-catenin activity during odontogenic differentiation of DPSCs. These data may help to guide DPSC behavior by tuning the N-cadherin-mediated cell–cell interactions, with implications for pulp regeneration.Dental pulp stem cell (DPSC) transplantation has shown new prospects in dental pulp regeneration, and is of great significance in the treatment of pulpitis and pulp necrosis. The fate and regenerative potential of stem cells are dependent, to a great extent, on their microenvironment, which is composed of various tissue components, cell populations, and soluble factors. N-cadherin-mediated cell–cell interaction has been implicated as an important factor in controlling the cell-fate commitment of mesenchymal stem cells. In this study, the effect of N-cadherin on odontogenic differentiation of DPSCs and the potential underlying mechanisms, both  Previouin vivo effect was examined using a subcutaneous transplantation mouse model.However, how cadherins regulate odontogenic differentiation of DPSCs remains largely unknown. Cadherins are composed of an extracellular domain that mediates calcium-dependent homophilic interactions between cadherin molecules, a transmembrane domain, and an intracellular domain . The int2 and 95% air. Cells cultured for 3–5 passages were used for the following experiments.Healthy dental pulp tissues were collected from caries-free teeth of patients (age 18–25 years old) undergoing extraction of fully erupted third molars, according to the informed protocol approved by the Ethics Committee of Nanfang Hospital, Southern Medical University, Guangzhou, China. Primary human DPSCs were isolated, identified and cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco), as described previously . The celTo induce odontogenic differentiation, the cells were incubated in odontogenic medium consisting of basal medium, 10 nM dexamethasone , 50 μg/mL ascorbic acid 2-phosphate (Sigma-Aldrich), and 10 mM β-glycerophosphate (Sigma-Aldrich) for the indicated times. The medium was changed every 2–3 days. For the β-catenin pathway inhibition experiment, cells were cultured with 2 μM XAV939 . Dimethyl sulfoxide (DMSO) (<0.2%) was used as for the vehicle-only control.TM RT Master Mix . Quantitative PCR analyses were performed using the LightCycler 480 Real-time PCR System  with SYBR® Premix Ex TaqTM (TaKaRa). The 2–ΔΔCt value was used to calculate the relative fold change of gene expression normalized to an internal control (β-actin). The primer sequences used were as follows:Total RNA was extracted from DPSCs using the TRIzol reagent  following the manufacturer’s instructions. Complementary DNA synthesis was performed using the PrimeScriptN-cadherin: forward 5′-TCTGGGTCTGTTTTATTACTCCTGG-3′, reverse 5′-GCGAGCTGATGACAAATAGCG-3′;Runx2: forward 5′-CACTGGCGCTGCAACAAGA-3′, reverse 5′-CATTCCGGAGCTCAGCAGAATAA-3′;DSPP: forward 5′-TCACAAGGGAGAAGGGAATG-3′, reverse 5′-TGCCATTTGCTGTGATGTTT-3′;β-actin: forward 5′-CCATCGTCCACCGCAAAT-3′, reverse 5′-CCTGTAACAACGCATCTCATA-3′.Total protein was extracted from DPSCs using RIPA buffer in the presence of protease inhibitor and phosphatase inhibitor  following the manufacturer’s instructions. The Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) was used in cases where the extraction of separate cytoplasmic and nuclear protein fractions was necessary. The Enhanced BCA Protein Assay Kit (Beyotime) was used to measure the protein concentrations. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes , and then incubated with primary antibodies overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The following primary antibodies were used: anti-N-cadherin , anti-Runx2 , anti-DSPP , anti-β-catenin , anti-Lamin B1 , and anti-β-actin . Proteins were visualized using enhanced chemiluminescence substrate. Protein levels were normalized to the β-actin or Lamin B1 signal.4 cells/well and were infected with viral supernatants (multiplicity of infection = 50) supplemented with polybrene (5 μg/mL) for 10 h, and then incubated under normal growth conditions for a further 72 h. After screening with puromycin (1 μg/mL), DPSCs stably expressing the N-cadherin-specific shRNA were established. The knockdown efficiency was confirmed by quantitative real-time polymerase chain reaction (qPCR) and western blotting.A lentivirus expressing a short hairpin RNA (shRNA) specific to the N-cadherin (oligo sequence: CCGGCCTAAG ATCATTCGCCAAGAACTCGAGTTCTTGGCGAATGATCTT AGGTTTTTG) and a negative control lentivirus (oligo sequence: CCGGTTCTCCGAACGTGTCACGTCTCGAGACGTGACACG TTCGGAGAATTTTTG) were provided by GeneChem . DPSCs were seeded into 6-well plates at a density of 6 × 10After culture in odontogenic medium for 7 days, DPSCs were fixed with 4% paraformaldehyde for 30 min at room temperature and washed with distilled water. Alkaline phosphatase (ALP) staining was performed with the BCIP/NBT ALP Color Development Kit (Beyotime) according to the manufacturer’s instructions. The total protein content was determined in the same sample by an ALP activity kit . The absorbance of each well was determined by measurements at 520 nm. ALP activity relative to the control treatment was calculated after normalization to the total protein content.After culture in odontogenic medium for 14 days, DPSCs were fixed with 4% paraformaldehyde for 30 min at room temperature and washed with distilled water. Calcium deposition in the ECM was stained with 2% Alizarin Red S (ARS)  for 15 min. The mineralized nodules were observed and photographed using an inversed microscope . To further quantify the mineralized nodules, the stain was dissolved in 10% cetylpyridinium chloride (Sigma-Aldrich) for 1 h, and the calcium concentration was determined by absorbance measurements at 562 nm.Dental pulp stem cells cultured on coverslips were fixed with 4% paraformaldehyde at 4°C for 15 min, and then permeabilized with 0.3% Triton X-100 for 5 min. After blocking with 10% normal goat serum for 1 h, the cells were incubated at 4°C overnight with anti-β-catenin  primary antibody. Subsequently, the cells were incubated at 37°C for 1 h in the dark with goat anti-rabbit conjugated with Alexa Fluor 647  secondary antibody. 4′,6-diamidino-2-phenylindole (DAPI) (Roche) was applied to visualize the nuclei. Images were captured using a fluorescent microscope (Olympus).Animal experiments were performed in accordance with the regulations of the Ethics Committee of Nanfang Hospital, Southern Medical University, Guangzhou, China. Five male BALB/c immunocompromised nude mice  were purchased from the Medical Laboratory Animal Center of Guangdong Province (China) and were maintained under pathogen-free conditions in the Experimental Animal Center at Nanfang Hospital, Southern Medical University.6 cells were loaded onto the β-TCP scaffold per block. The composites of β-TCP blocks and cells were transplanted into the left and right dorsal subcutaneous region of mice as previously described  blocks were provided by the Biological Materials Manufacturing Core, Sichuan University. DPSCs were transfected with either the control or N-cadherin-specific shRNA lentivirus, and a total of 1 × 10escribed . Four weThe embedded samples were serially sectioned at 4-μm thickness. The sections were deparaffinized in xylene, rehydrated through a series of graded ethanol solutions, and stained with hematoxylin and eosin (H&E) or Masson’s trichrome  according to the manufacturer’s instructions.The sections were dehydrated, subjected to antigen retrieval, and incubated with primary antibodies at 4°C overnight, followed by a horseradish peroxidase-conjugated secondary antibody for 40 min at room temperature. The immunostained proteins were visualized by the application of diaminobenzidine solution. The following primary antibodies were used: anti-DSPP , anti-nestin , and anti-β-catenin . The sections were lightly counterstained with hematoxylin, mounted, and analyzed under a light microscope (Olympus) by a technician who was blinded to the samples. The number of positive cells exhibiting brown staining were counted at 400× the original magnification in five randomly picked fields per slide, and the mean value of these fields was calculated for each group.F-test, respectively. Samples with normal distribution were analyzed with the independent sample t-test for two groups or one-way analysis of variance (ANOVA) followed by Bonferroni post-test for multiple groups. Samples without normal distribution were analyzed using the non-parametric Mann–Whitney U test for two groups or Kruskal–Wallis test for multiple groups. The statistical significance level was set at P < 0.05.All experiments were repeated at least thrice. Data are presented as the means ± SEM unless otherwise stated and were analyzed using SPSS software . Data normality and homogeneity of variances were confirmed with Shapiro–Wilk test and To explore the involvement of N-cadherin in the odontogenic differentiation of DPSCs, DPSCs were cultured in odontogenic medium for 3, 7, and 14 days, and then the expression pattern of N-cadherin and odontogenic markers (DSPP and Runx2) were examined by qPCR and western blot analysis. The expression of DSPP and Runx2 gradually increased over time as expected, whereas the expression of N-cadherin gradually decreased both at the mRNA and protein level . These rGiven that there is a negative correlation between N-cadherin and the odontogenic markers, it was investigated whether inhibition of N-cadherin could promote DPSC differentiation by knocking-down N-cadherin via lentiviral transfection . qPCR anNext, lentivirus-transfected DPSCs were subjected to odontogenic differentiation. Following induction for 7 and 14 days, ALP staining, alizarin red staining, qPCR, and western blotting were performed to identify the differentiation of DPSCs. Compared with the control shRNA lentivirus-transduced cells, the expression of odontogenic-related genes, DSPP and Runx2, were significantly upregulated in the N-cadherin shRNA lentivirus-transduced cells . Meanwhiβ-catenin is involved in both cadherin-mediated cell adhesion and the canonical Wnt/β-catenin pathway. To determine nucleus translocation and accumulation of β-catenin, a marker for β-catenin signaling activation was detected by western blotting and immunofluorescent staining. As shown in Further, lentivirus-transfected DPSCs were treated with XAV939, a specific inhibitor of β-catenin. As expected, XAV939 decreased the expression of nuclear, cytoplasmic, and total β-catenin, as well as odontogenic-related genes (DSPP and Runx2) . Consistin vivo, we performed subcutaneous transplantation with β-TCP/DPSCs composites in BALB/c immunocompromised nude mice for 4 weeks (To confirm the impact of N-cadherin on odontogenic differentiation of DPSCs  4 weeks . H&E sta 4 weeks . Masson’ 4 weeks . The lay 4 weeks . Similar 4 weeks . The num 4 weeks . These rin vitro, which was abrogated by pretreatment of the cells with XAV939, a specific inhibitor of β-catenin. In addition, knockdown of N-cadherin promoted the formation of odontoblast-like cells and collagenous matrix in β-TCP/DPSC composites transplanted into mice.The fundamental goal of endodontic treatment is to retain the natural dentition. Regeneration of a functional pulp-dentin complex is promising for the overall prognosis of the tooth . To regein vitro (in vitro and in vivo. Collectively, a functional decrease of N-cadherin expression appears to be a universal phenomenon during odontogenic/osteogenic differentiation. This may be explained by lessons learned from tooth development, since there are parallels between pulp development and regeneration (Mounting evidence shows that the mechanical and biochemical signals from cell–cell adhesion are key to stem cell fate decision . It was in vitro . In acconeration . During neration . Howeverneration . In thisneration .DANCR, regulate the odontogenic differentiation of DPSCs through the Wnt/β-catenin pathway (It is well-established that the role of N-cadherin involves both cell–cell interactions and interference with intracellular signaling, particularly the Wnt/β-catenin pathway. N-cadherin binds to β-catenin and modulates its cytoplasmic pools and transcriptional activity . In addi pathway .DSPP promoter (Moreover, we found that knockdown of N-cadherin increased the expression of odontogenic markers Runx2 and DSPP. Studies have illustrated that β-catenin binds to the Runx2 promoter and controls its transcription , and odopromoter . Hence, promoter . The staPulp regeneration, in its most strict sense, must include functional re-innervation of the pulp–dentin complex . InteresThe results presented here demonstrate that knockdown of N-cadherin enhances β-catenin activity possibly through release of β-catenin at the cell membrane and decreased interaction with the Wnt coreceptor LRP5/6, resulting in Runx2 and DSPP upregulation, and odontogenic differentiation of DPSCs . These dThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the Ethics Committee of Nanfang Hospital, Southern Medical University, Guangzhou, China. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics Committee of Nanfang Hospital, Southern Medical University, Guangzhou, China.ZD, WY performed the experiments, analyzed the data, and wrote the manuscript. XD, MC, and QQ participated in the experiments and revised the manuscript. WZ and BW designed the study and revised the manuscript. All authors have read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.