At the same time, microneedles can also provide mechanical support or directional traction for tissue, thus accelerating tissue repair. This review summarized the research progress of microneedles for in situ tissue regeneration over the past decade. At the same time, the shortcomings of existing researches, future research direction and clinical application prospect were also discussed.Tissue injury is a common clinical problem, which may cause great burden on patients' life. It is important to develop functional scaffolds to promote tissue repair and regeneration. Due to their unique composition and structure, microneedles have attracted extensive attention in various tissues regeneration, including skin wound, corneal injury, myocardial infarction, endometrial injury, and spinal cord injury et al. Microneedles with micro-needle structure can effectively penetrate the barriers of necrotic tissue or biofilm, therefore improving the bioavailability of drugs. The use of microneedles to deliver bioactive molecules, mesenchymal stem cells, and growth factors   This prex vivo systems, ISTR is relatively convenient and eliminates the need for harvested cells, making it more suitable for clinical translation [ISTR refers to the use of engineering biomaterials carrying biological active molecules  to direct endogenous progenitor or stem cells to repair damaged tissues ,14. Compnslation ,16. For nslation . Due to nslation . Angiogenslation , but shonslation ,21. To dnslation ,16. Amon2 to cm2) and micro-needles with a height of tens of microns to several nanometers [The MN is a novel drug delivery device consisting of a substrate  as a master mold . The matHydrogel MNs are made by cross-linking hydrophilic polymers such as poly(ethylene glycol) diacrylat (PEGDA) and gelatin methacrylamide (GelMA) ,65. The MNs can painlessly and minimally invasively penetrate a variety of tissues, including skin and cornea, without causing pain or inflammatory response ,70. Comp2.2in vivo.Due to the lack of penetration ability of conventional scaffolds such as hydrogels and sponges, necrotic tissue barriers or biofilms at damaged tissues may limit the permeability of active substances and lead to low bioavailability and poor therapeutic efficacy. Here, MNs with micro-needles structure provide an effective solution without using syringes which may cause inflammation and pain. For instance, by means of simple finger pressure, MNs can penetrate the skin tissue and deliver the active substance to the wound bed . And MNsin vitro stimulus such as near-infrared (NIR), intelligent MNs can be designed to control the release rate of active substances. Xia et al. encapsulated gelatin nanoparticles modified with the gelatinase-responsive antimicrobial photothermal peptide AMP-Cypate in dissolving MNs [S. aureus. Similarly, Wang et al. prepared NIR photothermally responsive NO-releasing MNs by embedding graphene oxide particles loaded with nitric oxide and metal organic frameworks into the porous structure of PEGDA hydrogel MNs [By utilizing the microenvironment of damaged tissue sites  or ving MNs . When apogel MNs . Zhao etogel MNs . Upon NIIt must be noted that due to the variability of mechanical strength and thickness of different tissues, the physical properties required for MNs to effectively deliver the active substance to the target tissue are different. For the design and preparation of MNs for tissue regeneration, the dimensions of the needles of MNs  are the first things worth considering. Due to the good tissue penetration of the conical structure , the neeOverall, recent researches have demonstrated the spatial precision of MNs for drug delivery while overcoming the complex and dynamic barriers of various tissues including eye and skin . Clinica2.3Due to the complexity of the tissue environment, additional demands are placed on the functions of MNs used for tissue repair. For example, MNs for bacterial infected wound repair require antimicrobial properties, pro-angiogenesis and tissue regenerative properties, and MNs for MI repair require pro-angiogenic activity and inhibition of myocardial fibrosis.Simply, multifunctional MNs can be obtained by incorporating multiple active substances into polymer solutions using a micromolding method. By this method, Professor Wang's group prepared HA MNs co-loaded with recombinant humanized type III collagen (rhCol III) and naproxen (Nap) encapsulated PLGA nanoparticles . After bThe second route is to apply the concept of coated MNs and prepare multilayer composite MNs to regulate the release rate of drugs. For example, as illustrated in Another approach is to embed other active substances in the base of MNs at the same time, which can effectively provide programmed treatment to damaged tissues . In addiInterestingly, when the needles and base of the MNs are made of materials with different dissolution or degradation rates, the drug release rate can be flexibly controlled. For example, a bilayer MN consisted of tetracycline hydrochloride (TCH) loaded HA needles, and deferoxamine (DFO) loaded chitosan and silk fibroin base. The TCH was completely released from HA needles in 25 min to promote early antimicrobial activity, while the release of DFO in the base was relatively slower . It is wFor dissolving MNs, encapsulating drugs in materials such as nanoparticles, micelles, and hydrogels that can release drugs continuously and slowly can better circumvent the negative effects of their explosive drug release and control the release rate of multiple drug combinations. Bilayer MNs prepared from a combination of dissolvable and hydrogel materials are an important research direction for the future. This is because they allow for increased drug loading and ease of use, while also intelligently controlling the rate of drug release according to the requirements of tissue repair process. Compared to direct doping method, bilayer MNs offer more flexible active substance combinations and easier control of release rates. However, the preparation process of bilayer MNs is more tedious and the reproducibility of the product may be less demanding. Considering the sequential nature of the tissue regeneration process, the on-demand release of active substances is particularly important. However, the preparation of MNs with drug release rates that can perfectly match the tissue healing process is still a considerable challenge due to the limitations of MNs preparation technology.2.4In addition to acting as a carrier of active substances, MNs, a minimally invasive medical device, can also modulate cellular behavior and provide a favorable environment for tissue repair. Luo et al. found silk fibroin (SF) MNs induced impairment of mechanical communication between fibroblasts and ECM, as well as reduced mechanical stress and contraction generated by fibroblasts . In addiMimicking nature's layered architecture and sophisticated strategies, bionic MNs with special structures are designed and manufactured to adhere tightly to tissue surfaces or generate traction to close wounds . For exaIn general, relevant studies have shown that MNs can provide physical stimulation, traction, and support to repair damaged tissues. However, the specific therapeutic mechanism remains to be further demonstrated. To date, most studies on MNs have focused on MNs as minimally invasive devices for drug delivery, and few have investigated the effects of the structure or materials of MNs on tissue repair. Surprisingly, with the development of micro and nano processing technology, some bionic MNs with specific structures have been designed and prepared for ISTR. These MNs have shown promising therapeutic effects and potential in areas such as wound closure, myocardial repair and hemostasis of the liver. However, the complexity of their preparation process also poses a challenge for their further research and development.33.1Skin wound care is a major social and economic burden, with millions of people suffering from poor wound healing each year worldwide. Approximately 1–2% of the population in developed countries is reported to suffer from chronic wounds . The nor2GeO4:Cu2+ (ZGC) into dissolving MNs [1O2, hydroxyl radicals and superoxide radicals) continuously over 48 h, effectively eliminate methicillin-resistant Staphylococcus aureus (MRSA) biofilms, reduce in vivo inflammation and promote wound healing. Similarly, antimicrobial agents such as water-soluble antibiotics  or antifloxacin , silver floxacin , and nanfloxacin ) have befloxacin ,114. Zhafloxacin . The preThe global prevalence of adult diabetes is expected to increase to 7.7% by 2030, and diabetic complications pose a serious threat to patients' health, leading to reduced quality of life, increased healthcare costs and mortality ,115. In MNs can effectively penetrate the skin, increase drug distribution to deeper tumor sites, and minimize leakage of therapeutic drugs to adjacent tissues, thus improving the therapeutic effect of cutaneous epidermal tumors . On the In conclusion, MNs can provide effective treatment for difficult-to-heal wounds such as bacterially infected wounds, diabetic wounds, and wounds caused by tumor resection. Considering the complexity of the wound physiological environment, bilayer MNs with flexible drug combinations and controlled drug release rates are one of the hot spots for future research. However, it is important to consider that the thickness of the skin layer varies from patient to patient, making the penetration depth of MNs potentially very different, leading to a wide range of abnormalities in the spatial distribution of active ingredients. Furthermore, most MNs for tissue regeneration are prepared by using fixed-shape molds. Despite the simplicity and reproducibility of the preparation process, also makes it difficult to provide effective treatment of irregular wounds with MNs. Although the shape and size of the mold can be personalized using image recognition , it also3.2The cornea, a multi-layered transparent tissue without blood vessels, is the most densely distributed nerve tissue in the body, with approximately 7000 injurious receptors per square millimeter . CornealBdellovibrio bacteriovorus (B. bacteriovorus) bacterial suspensions and freezing them at −80 °C  . IUA is 2) nanoenzymes and stem cells in the backing layer and tip of GelMA MNs, respectively [In order to scavenge ROS and improve the regenerative capacity of endometrium, Zhao et al. embedded antioxidant cerium oxide  with anti-inflammatory activity and antibacterial properties and human endometrium-derived epithelial cells (En-ADVs) . The stuGiven their unique structure and good biocompatibility, MNs can effectively deliver active substances to achieve endometrial regeneration while acting as a physical barrier to prevent tissue adhesion. However, there are relatively few studies on MNs for the treatment of IUA, and large-scale animal experiments and clinical trials are still needed to verify their effectiveness. Future research in the field of endometrial damage repair by MNs may favor flexible combinations between multiple active substances  and MNs, providing a wealth of options for the treatment of IUA. And it is important to consider the delivery system of MNs and the way they are inserted into the tissue when they are applied clinically to promote endometrial repair.3.4Ischemic cardiovascular diseases such as MI are the leading cause of death worldwide. MI is a process of adverse cardiac remodeling triggered by apoptosis and necrosis of myocardial cells, activation of inflammatory response, degradation of extracellular matrix (ECM) and tissue fibrosis following acute or chronic blockage of coronary arteries ,155,156.MNs can act as a channel between the patch and myocardial tissue. Prof. Cheng's group prepared PVA MNs carrying cardiac stromal cells . The MNsOn the other hand, MNs can also act as myocardial patches, providing mechanical support for heart to reduce myocardial stress and inhibit unfavorable ventricular remodeling . HoweverIn conclusion, MNs can promote MI repair by delivering active substances to infarcted myocardial tissue or providing mechanical support. MNs allow better integration of biomaterials and infarcted myocardial tissue than conventional patches. Tissue-adherent MNs can be delivered in a minimally invasive manner, thus avoiding suturing and open-heart surgery. However, most of the animal models are acute MI caused by ligation of the left anterior descending branch. In the future, more models are still needed to further validate the therapeutic effect of MNs. On the other hand, whether MNs can damage the normal tissue surrounding the infarcted myocardial tissue, destroy capillaries or cause inflammation still needs to be rigorously verified.3.5In addition to skin, cornea, endometrium, and myocardium, MNs have been also widely explored as controlled local delivery platform of active substances for the repair of damaged tissues such as spinal cord injury (SCI), stroke, and periodontal diseases. And relevant studies are summarized in 3.5.1in vivo results showed that local implantation of MNs significantly improved the retention rate of 3D-Exo, achieved slow release of active ingredients, and promoted the recovery of neural function. It is important to note that the mechanical properties of MNs should match those of the spinal cord in order to avoid causing tissue damage. In addition, the optimal time window for implantation of MNs remains to be further investigated.SCI is one of the most serious neurodegenerative diseases in modern society, causing sensory impairment and/or paraplegia in patients ,166. SCI3.5.2Periodontitis is a chronic inflammatory disease that leads to progressive destruction of periodontal tissue  and is the leading cause of tooth loss in adults . For var3.5.3in vivo results showed that MNs did not cause significant inflammatory response, and achieved successful transfection and uniform distribution in the cerebral cortex within three weeks after surgery. And the prepared MNs increased VEGF expression and enhanced functional angiogenesis and neurogenesis.Stroke is an ischemic or hemorrhagic disease mainly occurring in the microvascular system of the brain, resulting in cerebrovascular damage and irreversible damage to neurological function. And stroke is one of the major causes of death and severe disability worldwide ,180. Int3.5.4Electrical stimulation is a physical and convenient treatment that has been widely used to promote cell proliferation and tissue regeneration ,184. In 3.5.5ex vivo osteoarthritic human cartilage and in vivo rabbit osteochondral defects.Cartilage defects due to osteoarthritis are a serious tissue lesion for which effective treatment is still lacking . As a mi3.5.6Limited by the poor effectiveness of traditional clinical hemostatic techniques such as suturing, uncontrolled or excessive visceral bleeding caused by acute trauma is a common and serious medical problem, often resulting in patient death ,189. In 3.5.7The microscopic thickness of the outer and inner vessel membranes results in low bioavailability of traditional intravascular drug delivery methods such as balloons and stents . The higin situ delivery platform, MNs have demonstrated excellent ability to promote tissue regeneration in animal models of SCI, stroke, periodontitis and muscle injury. However, due to the lack of sufficient experimental data and clinical research support, the therapeutic effect of MNs needs to be further verified. At the same time, MNs are expected to be applied in more other tissues, bringing new opportunities for healing damaged tissues.As a novel 41.The micro-needle structure provides strong penetration to help biomaterials pierce the necrotic tissues, biofilms, vascular wall, corneal epithelial layer and other tissue barrier, thereby improving the drug bioavailability.2.Flexible combinations of materials  and structures (needle tips and bases) allow control of drug release rates, and drug spatial distribution on the tissues surface or deep layer, and damaged area or adjacent normal tissue.3.Regulating cell behavior by generating physical stimuli or providing engagement/adhesion for rapid wound closure.4.With their flexible preparation methods , material properties  and geometry, MNs can be delivered to the target tissue site by thumb press, vacuum pump, spring-loaded pen, balloon and catheter, etc., thus meeting the requirements of clinical applications.After more than ten years of exploration, a variety of multi-functional MNs represented by dissolving or hydrogel MNs have been developed for regeneration of various tissues, including skin, cornea, endometrium, myocardium, muscle, spinal cord, oral tissues and blood vessel. Among these, difficult-to-heal wound repair such as diabetic ulcers and bacterially infected wounds are currently the most extensively researched areas for MNs. In general, MNs offer several unique advantages over traditional scaffolds such as films, sponges and hydrogels.in vivo tissue repair performance of MNs was mainly evaluated in small animals such as mice, rats and rabbits. Due to their anatomical, physiological and biomechanical structure, they differ from humans. Therefore, human volunteers will need to be recruited in the future to test the therapeutic effects of MNs.Most of the current studies are using MNs as a device for drug delivery to improve delivery efficiency. But studies on the effects of the physical properties of MNs, including geometry, needle height, tip radius, needle density and material, on the tissue repair process are still lacking. As a drug delivery platform, the problem of low drug loading rate of MNs is still not well solved, therefore MNs require careful research on efficient drug delivery methods, intelligent drug release mechanism and pharmacokinetics. In addition, the clinicaltrials.gov) using the keywords ‘(microneedle) OR (microneedling) OR (micro needle) OR (microneedles)’ showed that more than 3/4 of the clinical trials are hollow or solid MNs in vaccination, dermatology, pain management, etc. And no clinical trials have been found on the use of MNs for ISTR. MNs used for ISTR have the ability to interact with and alter the microenvironment in vivo, resulting in the need for regulatory approval of all aspects of safety and performance . In addition, MNs have been relatively poorly studied in tissues other than difficult-to-heal wounds. As a result, more verification and validation tests for MNs used in ISTR require more effort and resources.Although MNs show promising potential in the field of tissue repair, unfortunately, most of the products currently on the market are for cosmetic and vaccination purposes. In addition, a search of the U.S. clinical trials database (The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Therefore, research and debates to date are ongoing in an attempt to find the most effective way to detect this analyte using this enzyme as the recognition center. The 3rd generation biosensors using direct electron transfer (DET) type enzymes are a great way towards practical devices. In this work, we developed a simple method for the functionalization of glucose oxidase with redoxable ferrocene groups in chloroform. The enzyme retained its activity after storage in this organic solvent and after the functionalization procedures. This enzyme functionalization strategy was employed to develop the biosensing monolayer-based platforms for the detection of glucose utilizing the quasi-DET mechanism. As a result of an electrochemical regeneration of the catalytic center, the formation of harmful H Aspergillus niger have commonly been used to determine blood glucose levels [Glucose is the main source of energy for the human body and brain. Abnormal glucose levels in the blood can lead to diabetic disease. Therefore, the development of an efficient glucose detection system still has paramount significance . Due to e levels . GOx is e levels . First-gResearchers are still debating whether this DET mechanism is possible in the case of GOx because flavin adenine dinucleotide (FAD) as a cofactor is deeply hidden inside this enzyme and DET is likely not possible ,4. HowevRecently, chemical tools for the site-selective functionalization of proteins opened a new area for a wide range of applications of this semi-synthetic protein conjugates towards biomedicine, bioimaging, and biosensing . Suzuki In this work, we used chloroform as an organic solvent for the functionalization of GOx with ferrocenyl aldehyde. Activities of this modified enzyme after storage in this organic solvent and after the functionalization procedures were tested and compared with data of the unaffected enzyme. We have also shown that these enzymes can be used as the active monolayers for the glucose 2.5th generation biosensors.Aspergillus niger of ≥100 U mg−1 (without added oxygen) and type I peroxidase from horseradish (HRP) of approximately 150 U mg−1 were purchased from Sigma Aldrich. All inorganic salts for the buffer solutions were purchased from Carl Roth GmbH + Co. KG. Ferrocenecarboxaldehyde, 4-mercaptobenzoic acid (MBA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma Aldrich. All the experiments were conducted using type II water (R > 18 MΩ) purified in a Milli-Q system; 50 mM pH 7.0 potassium phosphate buffer (PPB) solution was used.Organic solvents were purchased from ChemPur  and distilled prior to the experiments. The used lyophilized powder enzymes of type VII glucose oxidase (GOx) from −1) and in a wavelength range of 190–500 nm with a scan rate of 50 nm min−1 in a cell of 0.10 cm path length, the concentration of GOx solutions was 1.0 mg mL−1. The CD spectra were recorded two times and the results were averaged. Hydrodynamic diameters of our samples were measured using Zetasizer µV  and its compatible software. Prior to the experiment, the organic solvent was evaporated and the residue was dissolved in PPB buffer pH 7.0 solution. The solutions were filtered using membrane syringes (CA 0.45 μm) to remove large dust particles. The measurements were performed three times and averaged by the software. The morphology of the prepared surfaces was carried out by atomic force microscopy , and operated in tapping mode in the air. The pictures of AFM were visualized and analyzed using a Gwyddion 2.60 software.Electrochemistry of our samples was carried out using an EmStat3+ potentiostat PSTrace5 from PalmSens, Houten, The Netherlands. Ultraviolet–visible (UV–Vis) spectra were measured to evaluate the kinetic constants and for the structure characterization using an Evolution 300 Security UV–Vis Spectrophotometer . During the measurements, the samples were thermostated at 25 °C using an integrated thermostat. A JASCO J-815 Circular Dichroism spectrometer  was used to measure the circular dichroism (CD) spectra of the modified and native GOx in a buffer of pH 7.0. These CD spectra were carried out under a stream of Ar gas . ROTI®Spin, MINI-30 centrifuge tubes with molecular weight cut-off of 30 kDa were used to collect the ferrocene-modified GOx fraction on the filter of the tube. Samples of 0.5 mL were added to the test tubes and centrifuged for 7 min at 8000 rpm. The process was repeated until the entire volume of the samples (5 mL) was filtered. The collected precipitates were additionally washed with 0.5 mL of PPB. The supernatant was discarded and the collected precipitates were dissolved in 5 mL of PPB and used in further experiments.This enzyme was modified with ferrocenecarboxaldehyde (FcCHO). The experiment was carried out in chloroform, in which the o-dianisidine-peroxidase reaction employing UV–Vis spectroscopy. The obtained initial velocity (v) vs. the glucose concentration curves were used to calculate the Michael constants (MK), and maximal rate (maxv) values by applying the Michaelis–Menten Equation (1):C(GLU) is a concentration of glucose in the sample. These kinetic parameters (MK and maxv) for this reaction of the o-dianisidine oxidation were fitted by employing OriginPro 2015 software.Activities of the unaffected and modified enzyme samples were measured via the aid of a coupled −1, and then in 0.5 M H2SO4 solutions from −0.2 V to 1.75 V vs. Ag/AgCl using 40 scans at a scan rate of 300 mV s−1. The Au electrodes were washed with distilled water and then immersed in Eppendorf tubes containing 5 mM 4-mercaptobenzoic acid (MBA) dissolved in methanol and stored for one hour in a refrigerator at +4 °C. Subsequently, these Au/MBA electrodes were transferred to a tube containing a mixture of EDC and NHS solutions with concentrations of 2 mM and 5 mM, respectively. The Au/MBA electrodes were kept in this mixture for 30 min at room temperature. After this step, the electrodes were immersed in the Fc-GOx solutions (1 mg mL−1) in a buffer pH 7.0 solution. The electrode in this solution was tightly wrapped with parafilm tape and stored for 12 h in a refrigerator at +4 °C. For comparison, two control Au/MBA and Au/MBA/GOx electrodes were prepared under the same conditions but using only MBA and the native GOx, respectively.Gold electrodes were cleaned with Micropolish alumina 0.3 μm gel for 5 min, dipped in distilled water and stored in an ultrasonic bath for 5 min. Each electrode was cleaned by cyclovoltammetry (CV) in 50 mM KOH from 0 V to −2.6 V vs. Ag/AgCl using 40 scans at a scan rate of 300 mV s−1. Two scans were made per measurement and a second scan was used as the results. The experiments were performed by increasing the concentration of glucose C(GLU) until the signal stabilized (up to 30 mM). Similar measurements were made in an oxygen-free environment, i.e., the solution was purged with Ar gas for 15 min and the CV experiments were carried out under an Ar atmosphere. The limit of detection (LOD) for the corresponding electrode was calculated using the Equation (2):a is the slope and σ is a standard deviation of the linear curve and a signal to noise (S/N) ratio of 3.0 was used. These parameters were calculated using a Data analysis tool from MS 2007 Excel.For these CV measurements, a three-electrode system consisting of gold coated with the modified Fc-GOx or GOx monolayer (Fc-GOx or GOx/MBA/Au) electrode of a diameter of 3.0 mm as a working electrode, Pt wire of diameter of 1 mm as the counter and Ag/AgCl in saturated KCl as a reference electrode were used. They were immersed in 25 mL of PPB buffer pH 7.0 containing 0.1 M KCl to increase the electrical conductivity of the solution. For signal detection, a potential range from 0 mV to 600 mV was chosen at the scan rate of 20 mV s2O2 that reacts by catalyzing horseradish peroxidase (HRP) with o-dianisidine as the colorimetric probe at 460 nm. To estimate and compare Michaelis constant (MK) and maximal rate (maxv) as the catalytic parameters of the affected and unaffected GOx, a Michaelis–Menten model was used. The values of MK and maxv of these samples were measured to be 11.2 ± 0.8 mM−1, 6.0 ± 0.1 mM s−1, and 13.7 ± 1.6 mM−1, 5.2 ± 0.2 mM s−1 for unaffected GOx and after its exposure in CHCl3, respectively. These results show that the activity after GOx exposure in CHCl3 is lower but retains its catalytic properties. Therefore, this organic solvent can be used to functionalize this enzyme.To functionalize glucose oxidase (GOx) with a ferrocenyl group, ferrocenyl aldehyde as a hole mediator was used. However, this reagent is not soluble in water (PPB of pH 7.0 in our case). Therefore, this condensation reaction between free side amino groups of the lysine or arginine residues on the GOx surface and this aldehyde can only occur in organic solvents. Before performing the functionalization of the GOx surface, the enzyme stability after storage in chloroform as the organic solvent was tested using a coupled enzyme assay, in which GOx oxidizes glucose resulting in the production of HMK and maxv were measured to be 17.0 ± 1.1 mM and 4.5 ± 0.2 mM s−1 for Fc-GOx, respectively. The activity of Fc-GOx is lower than the unaffected GOx but Fc-GOx retains the catalytic properties.Compounds with the active aldehyde function groups have been used to functionalize the N-terminus residues of amino acids and for immobilization of enzymes onto electrode surfaces while maintaining the activity of these enzymes . Therefo3. The UV–Vis bands at around 330 nm and 450 nm should be characteristic of ferrocenyl aldehyde and its derivatives [n → π* transition in the aromatic amino acid moieties, with a charge transition from the flavin adenine dinucleotide (FAD) as the cofactor to indolyl moieties of tryptophane in the active center of GOx, and the full π → π* transition between the chain of the aromatic residues of the enzyme, respectively [3 was observed  method. 3. Moreover, the two fractions of nanoparticles with diameters of around 10 nm and 160 nm are characteristic of the Fc-GOx sample  as the classical amide bond forming and the immobilization reagents. These Au/MBA electrodes activated with the NHS groups were immersed into corresponding GOx or Fc-GOx solutions to obtain the SAMs of the enzymes (GOx/MBA/Au and Fc-GOx/MBA/Au) on the Au surface (Self-assembled molecular monolayers (SAMs) of DET-able enzymes have been applied as the best method for immobilization of these enzymes on the electrode surface because the tunneling distance is a key factor in DET-type bioelectrocatalysis . Therefo surface a.a), root mean square roughness (Rq) and maximum elevation were estimated by analyzing topography scans of these surfaces. The parameters of Ra, Rq, and maximum elevation were calculated to be 0.83 nm, 1.0 nm, and 7.4 nm for GOx/MBA/Au, 3.4 nm, 5.1 nm, and 43 nm for Fc-GOx/MBA/Au, and for comparison 0.66 nm, 0.93 nm, and 2.5 nm for bare Au, respectively. These AFM results demonstrate that the surface of GOx/MBA/Au is much less rough than Fc-GOx/MBA/Au because of structures, such as the aggregated enzymes with diameters of ~200 nm and highs of ~40 nm were detected on the Fc-GOx/MBA/Au surface. However, these structures on the surface are rare due to the relatively low concentration of nanoparticles in the solution used for immobilization. After AFM at the higher resolution of 200 × 200 nm, grains with diameters of ~10 nm and highs of ~2 nm were observed (−10 cm2 (200 × 200 nm). The concentration of the GOx molecules on the surface was calculated to be 3.5 × 1011 molecules cm−2 or 5.8 × 10−13 mol cm−2.To characterize the GOx/MBA/Au and Fc-GOx/MBA/Au surfaces, atomic force microscopy (AFM) was used. observed d. These observed e. The diobserved f. These observed c and froTo assess the suitability of the modified enzyme for use in biosensors, the electrode with the deposited Fc-GOx/MBA/Au monolayer was investigated and compared with the GOx/MBA/Au monolayer-based electrode. C(GLU)−1) for Fc-GOx/MBA/Au with and without oxygen are almost parallel. Therefore, the bioelectrocatalytic characteristics are the same with each other for Fc-GOx/MBA/Au with and without oxygen. Therefore, oxygen is not involved in this catalytic reaction when we used the functionalized GOx. However, a lower glucose concentration than around 0.1–0.2 mM cannot be detected by using the GOx/MBA/Au electrode. These values are equal to the concentration of oxygen in an air-saturated aqueous solution of ~0.2 mM. Similar results for carbazole derivatives as the hole transporting semiconductors coated electrodes were observed in our previous works [To show the bioelectrocatalytic properties of the electrodes, responses of the current densities to the concentrations of glucose at 300 mV vs. Ag/AgCl were depicted in us works ,9. There−2 mM−1 and 1.40 μA cm−2 mM−1 in the argon-saturated and aerated buffer, respectively. However, the lower values of the average sensitivities were estimated for GOx/MBA/Au under these conditions  at the applied potential of 300 mV vs. Ag/AgCl were calculated and summarized in These obtained characteristics of glucose sensing of the electrodes , entry 82. We have previously shown that direct hole and electron tunneling from the cofactor to the electrode (or vice versa) is not possible due to the long distance between the cofactor and the electrode surface. Secondly, the reduced cofactor regenerates by holes (H+) as the positive charge carriers from the side aromatic redoxable amino acid residues. These charge carriers come through these residues via the hole hopping mechanism from the mediator, which is nearest to the surface of the electrode. Finally, electron relays from the oxidized mediator  to the Au surface via the electron tunneling mechanism. Thus, an increase in the biocatalytic current is observed. Although without oxygen, this mechanism dominates, in absence of oxygen it competes with the classic mechanism, in which FADH2 is oxidized by oxygen to release H2O2. This reduced form of the cofactor can be oxidized by oxygen, so most biosensors of this type cannot detect concentrations below ~0.2 mM, because it is the concentration of dissolved oxygen in the aqueous solution. We have achieved that cofactor electrooxidation occurred without oxygen competition. Therefore, the search for new materials to help transfer holes from the enzyme surface to the cofactor is an important challenge.In this work, we show that electrical communication between a cofactor as the redox center of immobilized GOx and a gold electrode can be established by the chemical modification of the enzyme. This process is predominant in the absence of oxygen in the solution. In addition, we have previously predicted that hole hopping between the reduced cofactor and electrode surface through aromatic redoxable amino acid residues is possible . The pos11 molecules cm−2. The bioelectrocalatytic properties of the GOx and Fc-GOx-based monolayer-based electrodes were electrochemically investigated and utilized for the detection of glucose. The linear ranges from 20 μM to 80 μM with LODs of 5.2 μM and 8.3 μM (S/N = 3) for the Fc-GOx-based electrode were found in the argon-saturated and aerated buffer, respectively. However, glucose concentrations lower than ~0.2 mM cannot be detected by using the unaffected GOx-based electrode due to oxygen competition with the bioelectrocatalytic reaction. We have successfully demonstrated that the enzyme can be functionalized with the ferrocenyl groups in chloroform and applied for the detection of glucose via the quasi-DET mechanism. This quasi-DET ability of the functionalized GOx provides an opportunity to construct a mediator-free type glucose biosensor.In summary, chloroform is an excellent organic solvent to functionalize glucose oxidase with ferrocenyl as the redoxable groups. Using a coupled enzyme assay, we found that the activity of the functionalized enzyme (Fc-GOx) was lower than the unaffected GOx but Fc-GOx retained its catalytic properties. After our chemical manipulation in this organic solvent, diameters of this functionalized enzyme of ~10 nm and ~160 nm were observed from the DLS experiments, which correspond to individual enzymes and their aggregates caused by this organic solvent, respectively. The self-assembly monolayer of Fc-GOx was formed and characterized by AFM. These results have demonstrated the formation of a monolayer from the single-molecule enzyme layers on the gold electrode. Moreover, the concentration of the GOx molecules on this surface was calculated to be 3.5 × 10


The Hippo pathway consists of a cascade of kinases that controls the phosphorylation of the co-activators YAP/TAZ. When unphosphorylated, YAP and TAZ translocate into the nucleus, where they mainly bind to the TEAD transcription factor family and activate genes related to cell proliferation and survival. In this way, the inhibition of the Hippo pathway promotes cell survival, proliferation, and stemness fate. Another pathway can modulate these processes, namely the Wnt/β-catenin pathway that is indeed involved in cellular functions such as proliferation and cell survival, as well as apoptosis, growth, and cell renewal. Wnt signaling can act in a canonical or noncanonical way, depending on whether β-catenin is involved in the process. In this review, we will focus only on the canonical Wnt pathway. It has emerged that YAP/TAZ are components of the β-catenin destruction complex and that there is a close relationship between the Hippo pathway and the canonical Wnt pathway. Furthermore, recent data have shown that both of these pathways may play a role in neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, or Amyotrophic Lateral Sclerosis. Thus, this review analyzes the Hippo pathway and the Wnt pathway, their crosstalk, and their involvement in Huntington’s disease, as well as in other neurodegenerative disorders. Altogether, these data suggest possible therapeutic approaches targeting key players of these pathways. Altered homeostasis is observed in many neurological disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and Amyotrophic Lateral Sclerosis (ALS). Despite differences in etiologies, symptoms, and sites of pathology between these neurological disorders, a number of shared pathways have been identified for many or all of these diseases including Hippo and Wingless-Int (Wnt) pathways. These two signaling pathways play crucial roles in maintaining tissue homeostasis by orchestrating cell proliferation, differentiation, and apoptosis.The Hippo pathway consists of a cascade of kinases that finally controls the phosphorylation of Yes-associated protein (YAP) and a transcriptional coactivator with PDZ-binding motif (TAZ), hence their cellular localization. Unphosphorylated YAP and TAZ enter the nucleus and mainly interact with transcription factors known as TEA domain family members (TEAD) , promoting cell survival, proliferation, and stemness. It follows that YAP/TAZ, and, in general, the Hippo pathway, play crucial roles in various types of cancer, as widely demonstrated so far and explained in detail in several reviews ,3,4.2+ pathway [Wnt  is a family of glycoproteins that activate three different pathways: the canonical Wnt/β-catenin cascade, the noncanonical planar cell polarity (PCP) pathway, and the Wnt/Ca pathway ,6,7. In  pathway ,9.In HD, several symptomatic treatments have been used for decades, but many trials of potential modifier treatments focusing on neuron degeneration processes have failed ,11. MoreMany compounds that have been demonstrated to target the Wnt pathway are under investigation in clinical trials in AD, PD, and ALS , but notThe most important known functions of the Hippo pathway are to promote cell survival and proliferation but also differentiation and migration in developing organs ,14,15,16Besides YAP/TAZ, the most-studied coactivators of TEAD1–4 transcriptional activity are the Vestigial-like (VGLL) protein family, which consists of four members (VGLL1-4). These members interact with TEAD to regulate gene expression. VGLL1-4 bind to the YAP–TAZ binding domain of TEAD1-4, VGLL1-3 and VGLL4 have distinct molecular functions, and only VGLL4 has been reported to inhibit YAP/TAZ–TEAD interactions . The intThere are several upstream signals that regulate the Hippo pathway such as cell–cell contact, mechanical cues, and hormonal signals. For example, β-catenin is a linker between cadherins and the actin cytoskeleton that inhibits YAP activity in keratinocytes. The disturbance of the E-cadherin/β-catenin complex will then decrease YAP phosphorylation and consequently increase nuclear YAP localization ,40. The Regarding the regulation by circulating molecules, numerous studies have shown that the regulation of the Hippo pathway by GPCRs is a response of cells to hormonal cues and many GPCRs signal through YAP/TAZ ,47,48,49The Wnt/β-catenin signaling pathway is a cell survival pathway that is mostly involved in animal development, cell fate, cell proliferation, differentiation, and tissue regeneration and also has a central function in disease processes and tumorigenesis ,67,68,69Azzolin et al. demonstrated that YAP and TAZ are also components of the β-catenin destruction complex . YAP/TAZRecent studies have shown the deregulation of several actors of the Hippo pathway in HD. Mueller et al. observed that neuronal nuclear YAP levels are decreased in human post-mortem brains and neural stem cells (NSCs) derived from HD patients. Moreover, they showed that there is no change in YAP transcript levels in HD, suggesting that the decrease is due to post-transcriptional events such as phosphorylation. In accordance with this, they found a significant increase in phosphorylated YAP (pYAP) levels in the human HD cortex compared to the control but no change in the total YAP levels. In parallel, they also observed an increase in pMST1/2, despite no change in the total MST1/2 level; this probably explains the increase in YAP phosphorylation and its consequent decrease in the nucleus in HD post-mortem brains. Due to the decreased nuclear YAP, they observed a decrease in the YAP/TEAD interaction. Furthermore, in the post-mortem human cortex, they found three other downregulated Hippo pathway genes  as well as a downregulation of the Hippo target gene Cyr61. Mueller et al. also obtained similar results in Htt CAG knock-in mice HdhQ111/Q111, where they observed a significant increase in pYAP and in pMST1/2 levels .Similar results were obtained in another study by Yamanishi et al. They demonstrate that YAP is decreased in the nucleus of neurons in the cerebral cortex of human HD patients, and pLATS1 is increased in HD human neurons and mutant Htt-KI mice .Yamanishi et al. have found a novel form of necrotic cell death called Transcriptional Repression-Induced Atypical cell Death (TRIAD) in HD human and mouse brains. TRIAD is characterized by multiple steps: (i) the destruction of the cistern structures of ER, (ii) the enlargement and vacuolization of ER, (iii) the detachment of the outer nuclear membrane from the inner membrane, (iii) the release of ER content into the cytosol, (iv) the ballooning of the cell body, the shrinkage of the nucleus, and enucleation ,123. TheAll these results are very encouraging and have shown that acting on the Hippo pathway could represent an innovative therapeutic strategy to reduce HD phenotypes.Drosophila model [It is well established that the Wnt/β-catenin pathway is altered in HD models, especially the stability and levels of β-catenin. Studies have been performed in transfected cell models ,125, strla model ,128,129,Two studies show that the β-catenin level is decreased in HD. Firstly, Carmichael et al. observed a reduction in zβ-catenin level, associated with a reduction in TCF-mediated transcription in HD, and GSK-3 inhibition rescues polyglutamine-induced cell death in neuronal and non-neuronal cell lines (human neuroblastoma cells SKNSH and African green monkey kidney cells COS-7). The rescue is mediated by an increased level of β-catenin and its associated transcriptional pathway .Q111/Q111 striatal cells. This observation led to decreased β-catenin/TCF-mediated transcriptional activity with the downregulation of the Wnt/β-catenin target genes. Interestingly, the downregulation of β-catenin is not governed by GSK3β-dependent proteasomal degradation. Conversely, these authors observed an increase in miR-214 more than ten-fold in HD compared to controls; this miRNA was previously documented to decrease the β-catenin protein level in hepatocellular carcinoma cell lines [Ghotak et al.  found a ll lines ,131. Intll lines .Q111/Q111 striatum, Gines et al. observed an increase in Akt activation and signaling via the phosphorylation of GSK3β (which is inhibited), which led to decreased β-catenin degradation. In this context, they observed an increase in the levels of the β-catenin target gene cyclin D1 [In the same cellular model, as well as in extracts of Hdhyclin D1 .CAG140 and HdhQ111/Q111 knock-in HD mice, and post-mortem samples of HD patients. [Godin et al. firstly transfected MDCK and HEK293 cells with htt-480-17Q (expression of the N-term fragments of htt containing the first 480 aa with 17Q) or htt-480-68Q constructs. They observed a significant level of β-catenin with the htt-480-68Q construct with the two cell lines. β-catenin accumulated (in its phosphorylated form), but this accumulation does not lead to the transcriptional activation of the gene target. Then, they showed that the level of β-catenin is higher in different in vivo models of HD, such as drosophila, Hdhatients. .Two more recent studies conducted on induced pluripotent stem cell (iPSC) cultures confirmed these observations. Smith-Geater et al. showed, in HD neuronal cells derived from iPSCs, a significant dysregulation of several members of the pathway, including the downregulation of the components of the β-catenin destruction complex and the increased expression of Transcription Factor 3 (TF3) and frizzled transcripts, as well as the increased expression of Wnt transcriptional targets such as CCND1 . The uprIn conclusion, except for two publications ,127, it The Hippo signaling pathway has recently emerged as one of the important pathways playing a pivotal role in central nervous system diseases, including neurodegenerative disorders beyond HD, such as Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), some retinal degeneration disorders , cerebral ischemia-reperfusion, Alexander disease, dentatorubral–pallidoluysian atrophy (DRPLA), and Leber congenital amaurosis (LCA) ,133,134.Recent evidence also evoked the involvement of the Hippo pathway in Parkinson’s disease (PD). Ahn et al. demonstrated that in PD, there is a downregulation of Netrin1 (NTN1), which is a secreted laminin-related protein involved in nervous system development, axon guidance, and growth ,141,142.On the other hand, the Wnt pathway has also emerged to have an important role in neurodegenerative diseases, as came out from the constant increase in the literature on this topic. Indeed, numerous studies have shown that deregulations in the Wnt/β-catenin pathway are involved in the pathogenesis of neurodegenerative diseases such as AD, PD, and ALS. The Wnt/β-catenin pathway is known to have a neuroprotective effect against Aβ peptide in AD ,145,146,Similar evidence also emerged in PD. Mutations in the LRKK2 gene lead to the increased ability of LRRK2 to recruit GSK-3β and, as in AD, it phosphorylates the tau protein, leading to neurodegeneration . Indeed,Another neurodegenerative disease where the Wnt/β-catenin signaling pathway is involved is ALS. Studies in mice models demonstrated that several genes of the Wnt pathway, including ligands, receptors, co-receptors, and effectors are deregulated. It was observed that the upregulation of the Wnt1 ligand, Wnt3a, LRP5, and FZD receptors target genes such as Cyclin D1, c-myc, Fosl1, and Pitx2 ,170. LikTaken all together, these data show a strong relationship between the Wnt pathway and neurodegenerative diseases, and to deepen and go into detail on this relationship, it is useful to read Libro et al., 2016 and Serafino et al., 2020 ,174.All these data show the importance of the Hippo and Wnt pathways in neurodegenerative diseases. However, numerous studies are needed to fully understand the mechanism and role of these two pathways in neurodegenerative diseases, but the results are encouraging as they can represent a very important target in the treatment of these disorders.The activation of the YAP/TAZ–TEAD interaction can be achieved through two main mechanisms: (i) decrease YAP/TAZ phosphorylation and therefore increase their nuclear localization, (ii) favor the YAP/TAZ–TEAD interaction with TEAD ligands.The study of the overexpression of the representative members of different GPCR subgroups on YAP/TAZ phosphorylation by Yu et al. led to the discovery of the first activators and repressors of YAP/TAZ activity . LPA andA cell-based screening of about 18,600 molecules at Tokyo Medical and Dental University led to the discovery of TT-10 , an acti50 of 164 and 34 nM, respectively, by competing with ATP (10 µM). XMU-MP-1 . The discrepancy between the ATP concentration used in enzymatic assays and the cellular concentration of ATP explains the difference in power between the enzymatic inhibition and the cellular activity.To date, three inhibitors of Lats1/2 have recently been described. To compare the biological potency of these inhibitors, we indicate after IC50 of 0.2 nM (with 10 µM ATP) [TRULI  inhibits µM ATP) .50 of 0.76 nM  and 0.52 nM  [VT02956 has been identified through a high throughput screen of about 17,000 compounds in a program dedicated to identifying molecules for the treatment of breast cancer. Indeed, in this class of endocrine-resistant cancers, the expression of ESR1 (encoding ERα) is regulated by the expression of VGLL3, which recruits the NCOR2/SMRT repressor to the super-enhancer of the ESR1 gene. The inhibition of YAP and TAZ phosphorylation increases the nuclear import of YAP and TAZ, which compete with VGLL3. Finally, VGLL3 inhibits ESR1 expression. VT02956 in vitro inhibited LATS kinase with IC µM ATP) .50 of 4.1 nM  and 3.9 nM  [A cell-based, high-throughput screening developed for the identification of small molecules that promote cell proliferation (particularly in 3D conditions) led to GA-017 , a LATS  µM ATP) . This co µM ATP) . The autPobbati et al. reported the sole article on small TEAD ligands acting as activators of YAP/TAZ–TEAD rather than inhibitors . Q2 and Adihou et al. designed an eicosapeptide 4E   linked tFinally, recently, TRULI was used in an HD model of human neurulation to demonstrate that LATS could be a potential pharmacological target in HD .Several approaches have been studied to target the Wnt/β-catenin pathway in neurodegenerative diseases. Synthetic and biological molecules that are under preclinical studies or under investigation in clinical trials have been recently reviewed . The addThere is only one example of a drug that modified the Wnt pathway that was used in an HD model . The nonA new emerging druggable target is the carboxylesterase, Notum, which is responsible for the depalmitoleoylation of Wnt in the extracellular space . Its inhIn this review, we summarized the evidence that the Hippo and Wnt pathways are involved in Huntington’s disease. Post-mortem analysis of human HD brains showed the deregulation of several proteins of the Hippo pathway and particularly a decrease in nuclear YAP that correlates with the downregulation of the up-stream Hippo pathway regulators . SimilarAs seen above, the Hippo pathway and the Wnt pathway are both implicated in cell proliferation, survival, differentiation, and apoptosis. Literature analysis shows that these two pathways are studied individually, whereas the copious evidence of crosstalk between them should convince researchers to consider them together. The implications of each of these pathways have recently emerged in various neurodegenerative diseases, and new deregulations of these two pathways have been observed in these disorders. However, to date, no article has reported a study on the two pathways in the context of one neurodegenerative disease. It would be of great interest to have a precise idea of how these two pathways are connected in pathological tissues. Even if the design of drugs aimed at activating the YAP/TAZ–TEAD interaction (indirectly or directly) is still in its infancy, biologists now have efficient tool compounds to study the effects of therapeutic intervention in disease models. New drugs acting on the Wnt pathway are now available, and it would be interesting to have a precise vision of their effects on both β-catenin and YAP/TAZ pathways. Indeed, acting on the targets offered by both the Hippo and Wnt pathways has yielded positive results both in vitro and in vivo so far, and potential new targets and methods of action relevant to both pathways are emerging.Thus, new therapeutic approaches, including combination therapy, regarding these two pathways should be evaluated in the future, given the first promising results observed with some of these molecules described above.


The aim of the research was to estimate the effect of core–shell rubber (CSR) nanoparticles on the tensile properties, fracture toughness, and glass transition temperature of the epoxy and epoxy-based carbon fiber reinforced polymer (CFRP). Three additives containing CSR nanoparticles were used for the research resulting in a filler fraction of 2–6 wt.% in the epoxy resin. It was experimentally confirmed that the effect of the CSR nanoparticles on the tensile properties of the epoxy resin was notable, leading to a reduction of 10–20% in the tensile strength and elastic modulus and an increase of 60–108% in the fracture toughness for the highest filler fraction. The interlaminar fracture toughness of CFRP was maximally improved by 53% for ACE MX 960 at CSR content 4 wt.%. The glass transition temperature of the epoxy was gradually improved by 10–20 °C with the increase of CSR nanoparticles for all of the additives. A combination of rigid and soft particles could simultaneously enhance both the tensile properties and the fracture toughness, which cannot be achieved by the single-phase particles independently. Epoxy resins having relatively high tensile strength and modulus of elasticity, a low creep, and a good stability at elevated temperatures are extensively used as matrices in composite technology for different applications ,2. NeverTheir toughness could be improved by adding core–shell rubber (CSR) nanoparticles that are made of a soft rubbery core and a rigid shell around it which are mainly manufactured by emulsion polymerization and then added to the polymer resins. In comparison with the phase-separating rubbers, this method allows the advantage of controlling the particle size by changing the core and shell diameters . The matTg) [Tg and Poisson’s ratio and a significant decrease in the tensile and compressive properties of the CSR-modified composites which were explained by rubber having a lower Young’s modulus and a higher Poisson’s ratio in comparison with the epoxy [It was determined that the addition of CSR particles led to a significant reduction in the tensile properties of the epoxy resin (DGEBA) and almost no effect on its glass transition temperature (Tg) . For thehe epoxy . By usinhe epoxy ,7.In general, the fracture toughness of epoxy was improved by adding both rigid and soft particles ,9. The rThe research aimed to estimate the effect of core–shell rubber (CSR) particles on the tensile properties, fracture toughness and glass transition temperature of the epoxy and epoxy-based CFRP. The novelty of this work is in the multi-step approach for the evaluation of the toughening effects for both the epoxy and epoxy-based CFRP and considering their mechanical properties. The application of the proposed solution with improved fracture toughness both for the epoxy and epoxy-based CFRP could broaden their use in aerospace, automotive, marine and sporting goods due to them having a longer lifetime and enhanced safety features.CHS-Epoxy 582   was used3 [2 was supplied by Havel Composites  [Three additives containing CSR nanoparticles which were dispersed in DGEBA with different particle sizes and core material ACE MX 125, 156 and 960 were supplied by Kaneka . The information regarding core material and CSR size are given in epublic)  and usedFor pure epoxy samples, the epoxy resin was manually mixed with the hardener for approx. 10 min and the mixture was further degassed by using the vacuum pump. For CSR-modified epoxy resin, a certain content of CSR nanoparticles  was added to the epoxy and manually mixed, degassed, and then mixed with the hardener for approx. 10 min. After degassing, all of the mixtures were poured into silicon molds. The curing and post-curing conditions were chosen based on supplier recommendations [The silicon molds were used for the manufacturing of the test samples to determine the tensile properties ,15 and f12, which was cured at RT, cut into samples and post-cured as CSR-modified epoxy resin. The CSR nanoparticle fraction of 4 wt.% in the epoxy resin was used for the manufacturing of all of the CFRP plates based on the highest results of fracture toughness obtained for the modified epoxy in TDCB tests. At least five DCB samples were manufactured and tested for each CSR nanoparticle additive.Double cantilever beam (DCB) CFRP samples were produced by lay-up technology by using woven carbon fiber fabric (0/90)The morphology of the fracture surfaces for CFRP samples was examined by using a high-resolution SEM-FIB electron microscope Helios 5 UX , which was operated at 1 kV and 25 pA with scan interlacing and integration to avoid charging.For the test specimens of epoxy and epoxy that was modified with CSR nanoparticles, quasi-static tensile tests were performed by using Zwick 2.5 universal testing machine with a crosshead speed of 2 mm/min at RT. The tensile strength was defined as the maximal achieved value of stress in the specimen, and the elastic modulus was calculated from the slope of a secant line between 0.05 and 0.25% strain on a stress–strain plot. Five test samples per each CSR type and fraction were tested, and the values that are provided correspond to their arithmetic mean value.A specimen with a sharp pre-crack is needed for the precise measurement of the stress intensity factor (SIF). TDCB specimens produced in the silicone molds had an initial notch with a 1 mm width and a round end, which may substantially increase the apparent fracture toughness of the material. Therefore, the initial pre-crack of 2–5 mm length was made in the specimen before testing by the sharp knife strike. Moreover, side grooves of a depth of approx. 2 mm were produced to minimize the crack deflection and to keep the crack path along the midplane of the specimens . The tesPc is the critical load, b is the width of the specimen, and m is a geometrical parameter, which for the specimen of the considered geometry equals 0.6 mm−1. For the specimen with side grooves, Equation (2) should be modified asbn is the reduced width of the specimen at the grooves’ location, and the exponent value was determined from a series of 3D finite element simulations with grooves of different depths Kng=2pths see .3. Though this standard was specified for unidirectional laminates, it has been successfully applied for laminates with different lay-up configurations [GIC, was evaluated from the load–deflection curve at the point of deviation from linearity (NL). The NL calculation of GIC considers that the delamination starts to grow from the insert in the interior of the specimen at this point. The tests were performed by using Zwick 2.5 testing machine with a crosshead speed of 1 mm/min at RT and Canon EOS40D to record photos every 3 s for the analysis of the crack propagation until a failure occurred. ImageJ 1.38x software [The Mode I interlaminar fracture toughness tests of carbon 0/90 woven fabric laminates were carried out according to ASTM: D5528  using spurations . Accordisoftware  was usedP is the load, δ is the load point displacement, a is the delamination length, and Δ is determined experimentally by generating the least squares plot of the cube root of compliance as a function of delamination length.The Modified Beam Theory  method wF accounting for the shortening of the moment arm and the tilting of the end blocks and a displacement parameter N accounting for the stiffening of the specimen by the blocks—are recommended [L′ and t are the geometrical parameters of the blocks.Moreover, for the specimens with loading blocks, two correction parameters—a parameter ommended :(4)F=1−3Then, the corrected formula for interlaminar fracture toughness by using the Modified Beam Theory method takes the form:3. Then, the mass of the samples was registered in the air (ma) and the liquid of known density (lm). The density of the samples was determined by the formula:lρ and aρ are the densities of the liquid  and air (0.0012 g/cm3), respectively.The density of the epoxy and epoxy that was modified with CSR particles was defined at RT by using hydrostatic weighing in isopropyl alcohol and Mettler Toledo XS205DU balance with a precision of ±0.05 mg. First, the density of isopropyl alcohol was determined by using a sinker of a known volume of 10 cmTg) of the epoxy and epoxy modified by CSR particles was estimated by conducting thermomechanical analysis (TMA) tests using TMA/SDTA841e . The samples were heated from 30 to 150 °C at a heating rate of 3 °C/min and a force of 0.02 N, and then, they were subsequently cooled. According to ASTM standard E1545 [The glass transition temperature  and polymer matrix (epoxy), respectively, and while fv is the volume fraction of the filler, accordingly. The density of the epoxy was experimentally found to be 1.159 ± 0.002 g/cm3. Considering the known density of the 25%-CSR-modified epoxy of 1.1 g/cm3 [3 [The results that were obtained for the density of epoxy/CSR nanoparticle composites are shown in .1 g/cm3 , the denfc is the weight fraction of the filler.The volume fraction of filler could be evaluated as follows :(9)vf=ρmpρ is the density of the air and pv is the volume fraction of pores in the composites, respectively.As seen in The volume fraction of the pores can be derived from Equation (10):According to The stress–strain curves for the epoxy and epoxy that was filled with the ACE MX-156 CSR particles are given in Several analytical models, e.g., the Halpin–Tsai , Lewis–NfE and mE are the elastic moduli of the filler and the matrix, respectively.According to the Hansen model, the elastic modulus of the matrix that was filled with spherical particles was estimated by using the following formula:For the first step considering the epoxy matrix that was filled with the pores (air bubbles), Equation (12) becomes simplified since fE = 4 MPa [fv and pv, were evaluated from Equations (9) and (11), respectively.For the second step considering the epoxy matrix (with pores) that was filled with the CSR nanoparticles, Equation 12) was modified to include both pores and CSR nanoparticles was modiThe results of the evaluation by Equations (12) and (14) are shown in Tg for DGEBA type epoxy [Tg for the epoxy with the addition of the CSR nanoparticles. According to The results that were obtained for the glass transition temperature as evaluated using the TMA diagrams are provided in pe epoxy ,8,28. Cope epoxy , a reducpe epoxy ,4,21,28 The representative load–displacement curves for TDCB tests are provided in 1/2 which is slightly lower than the values that are reported in the literature for the epoxy resins [The fracture toughness of the epoxy which was evaluated by Equation (2) was 0.83 ± 0.07 MPa·my resins . In this2 to a maximal value of 599 ± 13 J/m2 as shown in The typical DCB load vs. displacement curves of the unmodified and CSR-modified CFRP laminate specimens are shown in However, the toughening effect of the CSR nanoparticles in the epoxy did not fully transfer to the epoxy-based CFRP composite laminates. E.g., the use of an epoxy that was modified with 4 wt.% of ACE-MX 156 having the maximal improvement of fracture toughness by 108% as a matrix for CFRP laminates resulted in the improvement to the interlaminar fracture toughness by only 32%. The interlaminar fracture toughness was maximally improved by 53% for ACE MX-960 at CSR content 4 wt.%. A further increase in the CSR fraction could result in greater improvement of the interlaminar fracture toughness of the CFRP, though, it should be emphasized that rubber toughening has also the side effect of increasing the viscosity of the epoxy resin, thereby negatively contributing to the fabrication of composite laminates ,28. AddiThe epoxy resin was modified by the addition of three types of CSR nanoparticles of different contents. On the one hand, the addition of all of the additives containing the soft CSR nanoparticles resulted in a minor decrease in the density, and a substantial reduction in the elastic modulus and tensile strength of the epoxy resin. The Hansen model was applied to describe the elastic modulus of the epoxy having a certain fraction of the CSR nanoparticles and pores, and a good agreement with the experimental results was found at the high CSR contents.On the other hand, it was testified that the fracture toughness of the epoxy was significantly improved by the addition of all of the investigated types of CSR. The optimum CSR nanoparticle content was found to be 4 wt.% for all of the CSR nanoparticle types, thereby resulting in the improvement of the fracture toughness of the epoxy by 60–108%. No considerable distinction in the fracture toughness among the additives was detected, thereby proving that the small (100 nm) and large (300 nm) CSR nanoparticles were equally efficient.Moreover, the effect of all of the additives containing the CSR nanoparticles was substantial, leading to the improvement in the interlaminar fracture toughness of the CFRP by 32–53%. Although, the toughening effect of the CSR nanoparticles in the epoxy was two times higher than it was in the epoxy-based CFRP composite laminates.Additionally, a gradual increase of the glass transition temperature was obtained for the epoxy that was filled with all of the additives containing CSR nanoparticles, which could be attributed to the high crosslink density and toughening effect of rubber modifiers, thereby testifying to their dissolution in the epoxy continuous phase.The possible combination of rigid and soft particles could be a compromise to simultaneously improve both the tensile properties and the fracture toughness, which cannot be achieved by the single-phase particles independently.


Generation of motor neurons (MNs) from human-induced pluripotent stem cells (hiPSCs) overcomes the limited access to human brain tissues and provides an unprecedent approach for modeling MN-related diseases. In this review, we discuss the recent progression in understanding the regulatory mechanisms of MN differentiation and their applications in the generation of MNs from hiPSCs, with a particular focus on two approaches: induction by small molecules and induction by lentiviral delivery of transcription factors. At each induction stage, different culture media and supplements, typical growth conditions and cellular morphology, and specific markers for validation of cell identity and quality control are specifically discussed. Both approaches can generate functional MNs. Currently, the major challenges in modeling neurological diseases using iPSC-derived neurons are: obtaining neurons with high purity and yield; long-term neuron culture to reach full maturation; and how to culture neurons more physiologically to maximize relevance to in vivo conditions. Movement disorders are a group of neurological conditions that cause either increased or decreased or slow movements. The movements may be voluntary or involuntary, and implicated in many neurological diseases, such as Dystonia, amyotrophic lateral sclerosis (ALS), Ataxia, Parkinson’s disease , and so iPSC-based disease modeling starts from Yamanaka and his colleagues’ groundbreaking studies, which demonstrated that somatic cells could be reprogrammed into pluripotent stem cells by ectopic expression of four transcription factors (Oct4 (O), Sox2 (S), Klf4 (K), and c-Myc (M)) under defined culture conditions ,10. An eIt is also well known that iPSCs and iPSC-derived cells have limitations, especially in the context of culture heterogeneity and dosage variability. The best route of administration and survivability in the hostile inflammatory microenvironment is controversial . ImproveOn the other hand, iPSC-based disease modelling is relatively new in biomedical research. Substantial progress has been made in developing differentiation protocols for the generation of different cell types. Excitingly, some in vitro models can recapitulate aspects of neuronal synaptic networks, which greatly advances functional modeling of neurodevelopmental and neurodegenerative diseases. Recent studies generating iPSC-derived MNs from movement disorders of ALS ,37,38 anMany studies are attempting to generate mature MNs from hiPSCs for modeling movement disorders. Developmental biologists have identified the signaling molecules and transcription factors that are involved in MN differentiation and maturation, providing the foundation for the generation of hiPSC-MNs. Currently, scientists are using these extrinsic factors to guide the MN differentiation from stem cells, thereby mimicking regionalization processes during nervous system development. Many protocols have been developed that rely upon core signaling pathways, which could synchronize neuronal induction to MN-specific signaling cascades and upregulate the expression of MN-specific genes.The generation of hiPSC-MNs is a consecutive process that consists of a variety of induction stages, including iPSC induction, embryoid body (EB) formation, neural rosette growth, neuron progenitor cells (NPCs) differentiation, and MN induction and maturation A. At eacHuman nervous system development begins with neural induction converting ectodermal cells into neuroectodermal cells, leading to the formation of the neural plate and finally the neural tube ,41. How One of the many ways of generating different types of neurons is through the differentiation of NPCs, which are a homogenous, multipotent, undifferentiated, self-renewable cell population. NPCs are committed to become neural lineage and to be differentiated to specific neuronal types in defined culture conditions . iPSCs aRecently, two approaches were reported for generation of NPCs using a combination of small molecules: either RA and VPA  or LDN-193189 (SMAD inhibitor) and SB431542 (Activin/BMP/TGF-beta Pathway Inhibitor) ,77,78. BSmall molecules are bioactive compounds that can modulate specific cellular pathways involved in cell signaling, transcription, metabolism, or epigenetics, all of which are modulated during cellular reprogramming. If selective epigenetic modulation can be achieved with chemicals, it could remodel the chromatin structure and activate the gene expression of transcription factors, achieving similar effects to the ectopic expression of reprogramming factors. The advantage of using small molecules in reprogramming is that their biological effects are typically rapid, reversible, and dose-dependent, allowing precise control over specific outcomes by fine-tuning their concentrations and combinations. In To acquire the MN progenitor identity, NPCs need to caudalize and ventralize with the action of RA and Shh, respectively ,109. ShhSmall molecules have a profound influence on neural induction and promote hiPSC differentiation into MNs combined with the simultaneous inhibition of TGFβ activin, Nodal, and BMP  signaling. Dual SMAD inhibition is a well-established method which utilizes small molecules to block endodermal and mesodermal cell fates and promote neuroectoderm conversion. It dramatically enriches neural ectoderm from pluripotent cells with a high expression of PAX6 and sex-determining region Y-box 2 (SOX2). Noggin and SB431542 are the most commonly used dual SMAD inhibitory small molecules. Noggin acts as a BMP inhibitor and SB431542 inhibits the Lefty/Activin/TGFβ pathways by blocking the phosphorylation of ALK4, ALK5, and ALK7 receptors. The GSK-3β inhibitor promotes neural progenitor proliferation by stimulating the canonical Wnt signaling pathway, which contributes to the maintenance of neural precursors. Molecular activation of SHH by Purmorphamine, canonical WNT signaling, and neural patterning by RA have been critical for MN induction. Previous studies have found that Shh induces upregulation of transcription factors OLIG2, NK2 homeobox 2 (NKX2.2), and neurogenin2 (NGN2) to direct the expression of MN fate-consolidating genes such as HB9 and ISL1. Neurotrophic and growth factors, such as BDNF, GDNF, and NT3 are also used as supplements to facilitate MN growth, maturation, and survival ,29,126. Scientists have been working continuously to advance our understanding of MN differentiation and to improve the techniques for generating MNs from iPSCs. The rapid advancement of RNA sequencing technologies contributed to a deep understanding of transcriptome composition and has discovered a large number of non-coding RNAs (ncRNAs) that participate in MN differentiation . These nMature miRNAs are ∼22-nucleotide single-stranded RNAs that can recognize the 3′ untranslated region (UTR) of its target mRNAs and negatively regulate gene expression post-transcriptionally ,147,148.miR-218 is the most abundant and highly enriched miRNA in developing and maturing MNs ,137,158.Chen and colleagues demonstrated that the repression of Olig2 mRNA (MN progenitor marker) is controlled by mir-17-3p microRNA ,150. TheLong non-coding RNAs (lncRNAs) are RNAs that exceed 200 nucleotides in length, and they are not translated into proteins. LncRNAs participate in various stages during MNs differentiation, including guiding neural fate choice by driving transcription factor localization ,165,166,The lncRNA CAT7 (chromatin-associated transcript 7) is a polyadenylated lncRNA that lies upstream (~400 kb) of MNX1 (MN homobox1). CAT7 has been found to temporally regulate MNX1 expression during the early stages of human ESC-MN differentiation. Loss of CAT7 causes de-repression of MNX1 before committing to motor neuronal lineage . AnotherNEAT1 (nuclear-enriched abundant transcript 1) is a well-characterized lncRNA that functions as a chromatin regulator and organizes nuclear structures called ‘paraspeckles’. Paraspeckles contain proteins involved in transcription and RNA processing ,175. NEAPrevious reports have underscored the essential roles of transcription factors in MN development . DecadesTranscription factors ISL1 and LHX3 are sufficient to activate a MN gene expression program in other neural progenitors and increase post-mitotic specification by directly reprogramming pluripotent stem cells into MNs ,189,190.The combination of different transcription factors was often used to obtain a high quality of iPSC-MNs. These transcription factors need to be delivered into cells via lentiviral vectors or other vehicles . In 2013Goparaju et al. showed that overexpression of NGN1 (Neurogenin 1), NGN2 (Neurogenin 2), NGN3 (Neurogenin 3), NEUROD1 , and NEUROD2  in human PSCs combined with RA, forskolin, and dual SMAD inhibition via SB431542 and dorsomorphin yields highly pure neuronal cultures expressing the MN markers HB9, ISL1, and ChAT . MNs genTo ensure that the high quality and purity of MNs can be obtained from hiPSCs, it is necessary to validate the cell identity at each stage during the process of induction and differentiation. Specific markers at different stages could be examined and used to estimate the induction quality and the MN purity B. As thePatient-specific somatic cells, such as skin fibroblast cells and peripheral blood mononuclear cells (PBMC), can be reprogrammed into the pluripotent state through ectopic expression of Yamanaka factors . The iPSC must be fully characterized to ensure quality before differentiation into neurons or other cell types. The morphology of iPSCs should demonstrate a typical hESC-like appearance composed of tightly packed cells in phase contrast microscopy B. A healTo achieve more accurate disease modeling and maximize the potential applications, quality controls are critical to verify cell identity and purity. Many methods could be used, including specific marker expression, molecular and functional properties, cellular morphology assay, electrophysiological analysis, and animal transplantation. Many protocols analyze samples within 2 to 5 weeks from the onset of differentiation. Early stages of MNs showed the typical polygonal cell body with few and short dendrites (Weeks 1–2). However, the generation of ISL1/2 and HB9-expressing cells can vary from 3 days to 2 weeks after differentiation. Longer differentiation protocols have an advantage over shorter ones when preparing fully functional mature neurons, which could be used to examine the electrophysiological activity and synaptic network. After treatment with lentivirus-expressing-specific transcription factors or small molecules, usually within a week the cell will become neuron-like with condensed nuclei, long axons, and multiple neurites. Generation of MNs requires the identification of genes that are expressed at the initial stages of MN differentiation. At day 13 of differentiation, studies observed the expression of early MN-specific factors, PAX6, OLIG2, ISL-1 and NEUROD . PAX6 anAs HB9 is downregulated over the course of MN maturation, the expression of choline acetyltransferase (ChAT) appeared and temporally increased . As ChATThe final maturation can be achieved with the action of neurotrophic factors  and demonstrated by increased dendrites arborization and cell-cell connections (weeks 2–6). Mature MNs display larger soma, increased cell shape, complexity of neurite outgrowth, and electrophysiological properties. iPSC-derived MNs are generally considered mature after 3 weeks of differentiation . Higher Modeling MN-related diseases using hiPSC-based approaches requires culture conditions in a dish that can recapitulate the events underlying MN differentiation, maturation, aging, and degeneration. Several protocols exist to generate MNs from hiPSCs, and these cells have been used to study the pathophysiology of MN-related diseases, such as ALS, spinal muscular atrophy, and DYT1 dystonia ,217,218.Generation of hiPSC-MNs overcomes the limited access to human brain tissues and provides an unprecedented approach for modeling MN-related diseases, offering an excellent platform for developing therapeutic treatments. However, several challenges arise from using this cellular system for disease modeling . First, To resolve these issues, studies are needed to understand the precise regulatory mechanisms of neural differentiation and maturation using in vivo models. Meanwhile, the protocols for the generation of iPSC-derived neurons need to be updated, simplified, and finally standardized to obtain consistent outcomes for biomedical research. The generation of different neuronal subtypes requires different protocols consisting of different induction factors, specific culture conditions and supplements, and particular treatments and processes. Generally, induction efficiency and neuron survival are paramount to achieve good yield and purity. The combinations of small molecules and/or transcription factors will enhance the induction efficiency and maximize the purity, while the optimized culture conditions will promote neuron maturation and survival, which will lead to a higher yield. One major difficulty in obtaining fully mature iPSC-MNs is the long-term culture issue, in which both neuron survival and potential contaminations are huge challenges. We generate iPSC-MNs using lentiviral delivery transcription factors, and these MNs can reach full maturation at 3 wpi with characterization of high expression of presynaptic proteins, cholinergic markers, and firing action potentials. For setting up experiments that require fully mature neurons, we usually culture iPSC-MN for 4 wpi . For suc