The current scenario of the quest for microneedles (MNs) with biodegradability and biocompatibility properties is a potential research area of interest. Microneedles are considered to be robust, can penetrate the skin’s deep-seated layers, and are easy to manufacture, and their applications from the clinical perspective are still ongoing with standard escalation. This review paper focuses on some of the pivotal variants of polymeric microneedles which are specifically dissolvable and swell-based MNs. It further explores the drug dissolution kinetics and insertion behavior mechanisms with an emphasis on the need for mathematical modeling of MNs. This review further evaluates the multifarious fabrication methods, with an update on the advances in the fabrication of polymeric MNs, the choice of materials used for the fabrication, the challenges in polymeric MN fabrication, and the prospects of polymeric MNs with applications pertinent to healthcare, by exclusively focusing on the procurable literature over the last decade. Continuous efforts and investigations are underway to develop a substitutional approach to the oral consumption of drugs and medications, for which the conventional method is hypodermic injections. The idea of the transportation of drugs through transdermal routes, wherein the pharmaceutics are transported to the skin’s innermost layers, was conceptualized and has garnered significant interest from the medical community . One of The transdermal means of transporting drugs through gels, ointments, and patches also have some hindrances in the delivery of their applications . ExpliciIn this regard, MNs are inexpensive, economical, and have a self-administrating type of TDD system for the delivery of various pharmaceutical drugs. They tend to permeate the stratum corneum (SC) to form transient microchannels that can dynamically diffuse micro-sized foreign molecules via the bloodstream . MNs canPolymeric MNs’ capacity is expected to hike up as research on TDD in conjunction with polymeric MNs and their usage gains momentum. However, several challenges need to be addressed before these MNs can be used extensively in medical applications. Irritability of the skin, contamination on the micro-level and a lack of mechanical ruggedness in biomaterials, the volume of drug to be loaded on and in the MN, and the delivery of hydrophilic macromolecules are all challenges that need to be catered to . ConsequThere is a significant need to cater to some of the primary and essential issues, such as the transformation from lab-based to user-based MNs, and their advancements towards product commercialization. This comprehensive-based review aims to discuss the latest techniques and developments related to the trials and challenges encountered by polymeric MNs in the areas of drug delivery routing mechanisms, polymer to polymer-based kinetics, type of materials used, and their variants with their evaluation techniques. Since the last decade there has been a sharp rise in the publications based on polymeric MNs, which implies the potential need for microneedles that are manufactured using polymers, which are graphically represented in Polymeric MNs have demonstrated their supremacy over silicone, metal, and other micro needles . They aiThe choice of material for the MN manufacturing and properties of kinetics for the drug release will play a pivotal role in the transformation of MNs into commercialized applications for effective treatments for various ailments. The Georgia Institute of Technology, USA, and Alza Company, USA, have carried out preliminary pilot studies concerning the progress of MNs for the most effective medical treatments in the early period of the 1990s. Conventional metals or silicone were used in the early years as an opted-for material to manufacture of MNs . As timeKinetics is the study of the rate of change in levels of concentration. Dissolvability or dissolution means the course by which a substance alters its form from a solid state to an aqueous solution over time. Thus, dissolution kinetics is the study of the rate at which a substance’s concentration changes as it dissolves . As a re3 (sodium bicarbonate), which is contained inside the microspheres. As an outcome, CO2 was formed, causing pressure to build up within the microsphere. The pressure was applied to the microsphere membrane until it was ruptured, allowing the encapsulated drugs to be released.The term pH-responsive polymers refer to polymers that can allow a substance to be transferred into acidic or alkaline bases. Hollow-shaped MN arrays with microspheres encapsulating two pharmaceutical drugs along with sodium bicarbonate were produced by Ke et al. . ProtonsMoisture responsiveness is a term used to describe a material’s ability to absorb moisture. Polymers that are reacting for moisture responsivity dissolve due to the effect of hydrolysis. The moisture-responsive-based dissolving type of MNs can be designed either for an immediate or for a controlled release mannerism of a drug . Koh et Enzyme responsivity of microneedles refers to the mode of sensitivity of the embedded drug in microneedles. Any variations or alterations in the biological signal are captured by these microneedles through application-oriented investigations and diagnostic purposes. The concerned pathologists and research community take advantage of the changes in the physiological signals by considering the parameters such as the overrated enzyme-based expressions, which ultimately trigger the release of the drug to the specific location.In patients with diabetes, suffering from fluctuating blood glucose levels is faced and there is a dire need for them to keep themselves regularly monitored and cautioned about the baneful effects. In order to follow that, they must be administered with anti-diabetic medicines or anti-diabetic drugs through an injection. Until today, the most convenient and pain-free method of insulin administration is carried out with the use of glucose-responsive-based MNs. Glucose-sensing devices which follow the methods are discussed below ,44,45.2) and consumption of oxygen (O2) and thereby generate hydrogen peroxide H2O2 [Enzyme catalyzed reaction depends on the content of the glucose oxidase (GOx) as a primary catalytic enzyme primarily used for conversion of glucose to gluconic acid. The reaction will result in a substantial decrease in the potential of hydrogen-responsive-based devices are also prepared using other different kinds of materials known as lectins. The most common lectin which is widely in use is concanavalin A, which is a tetravalent-binding protein.Glucose(CMolecular-based recognition with the help of binding chemical moieties ,45.MNs are miniaturized versions of the minimally invasive drug delivery type of devices, which necessitate an accurate mannerism and recurrent injection pricks on and into human skin layers . Lutton Material choices are hypercritical, and they should be capable of controlling the manner of drug release dynamics and their stability during manufacturing for a safe and effective usage of MNs ,48,49. TChen et al.  revealedIt is therefore understood that widening the MN bases, which simultaneously reduces the aspect ratio, would make skin insertion more challenging . SharpenA wide range of factors influence the skin thickness, including the age, gender, ethnicity of the individual, body extent, and also hormonal status . When MNBecause the thickness of the epidermis can reach up to 1500 m and MN lengths can reach up to a level of 1500 m, the drug is efficiently released into the epidermis. Most MNs range from 150 m to 1500 m long with a width from 50 to 250 m, having a thickness of the tip ranging from 1 m to 25 m . MNs witBy adjusting the height of the MNs meticulously, the drug dosage can be precisely administered, which results in good drug distribution in the skin without any wastage ,69. MN pThe main goal of mathematical modeling is to achieve the optimal shape or desired design for microneedle optimization ,73. The However, Sandrakov et al.  demonstrGomaa et al.  showed tKim et al.  depictedRonnander et al.  establisScientific and technological advancements have aided in the rapid production of versatile MN manufacturing techniques in recent years. The micro-molding method is considered to be an ideal fabrication method for manufacturing polymer-based MNs due to its high replicability, ability to easily scale up the production, and cost-effectiveness , with thAir, when being used directly for solidification of droplet-based polymers, helps in the development of the microneedle shape . This drThe procedure of drawing lithography involves creating microstructures that vary from a three-dimensional form to two-dimensional forms . Above aThis technology helps in achieving models according to the required geometrical shapes and sizes with the support of computer-aided designs ,95,96. TEmulsion techniques produce biodegradable microspheres, which can be easily be poured into a mold of MNs to form porous structures ,101. PLAStep 1: The prepared PLA microspheres is poured into a female PDMS mold.Step 2: It is pushed to the female-based cavities by the male structure of the PDMS.Step 3: The pushing and casting process is repeated till the mold is filled with the microspheres.Step 4: Metal plate is commonly placed at the base of the mold and the top with a sheet of PDMS.Step 5: The tip is pressed with the PDMS-based mold and ultrasonically welded. Heat is thereby generated when there is a friction with the microspheres, which ultimately yields a bond .Porous-based MNs with bio-ceramic materials are fabricated using these micro-molding fabrication techniques:Step 1: Porous structures are prepared, and acidic-based solutions are blended thoroughly.Step 2: The blended mixture is poured, then vacuumized, and finally cured in a female mold at a temperature of approximately 37 °C for two days.Step 3: The MNs, which are interconnected pores, are produced by mixing water and acidic solutions, and they are cured at ideal conditions. The primary advantage of this fabrication technique is that the drug can be directly blended with the ceramic-paste-based materials and can be loaded onto the whole MN female mold . Figure Even if the polymeric MNs are found to be biocompatible under the assumption of polymeric material accretion in the human or animal bodies when they are injected, they may cause impairment issues in the hepatic system accompanied by some detrimental immune-based reactions when these materials are inserted. To mitigate this, the insertion capabilities of the polymeric MNs need to be strengthened to make sure that they will not suffer due to breakage and bending issues after being inserted into the skin tissues .To tackle the challenge, an amalgamation of a minimum of two or more polymeric materials is to be prepared to alleviate the structural dynamics and mechanical robustness of the polymeric MN  simultanSeveral studies have been conducted through in vivo means of fabrication of MNs, which is used to transport pharmaceutical drugs and vaccines. The daunting task is to fabricate the MN, which can efficiently transport the macro-sized molecules with good molecular loads and a high amount of hydrophilicity ,104,105.As discussed previously, the concept of 3D printing is being exercised vigorously by the research community and pharmaceutical and biotechnology-based industries. The usage of this technology stands out as promising when compared to that of the conventional methods of manufacturing, in terms of the amount of time consumed for manufacturing and cost-effectiveness ,107,108.The microneedle found its way to tackle the global pandemic issue of COVID-19. The research team—Chen et al.—worked out and presented MN-based oropharyngeal swabs which substantially reduced the false negatives in testing procedures . This coIn the last decade, MNs have garnered keen interest from the research and industrial expert communities for their advantages in the area of transdermal drug delivery-based applications, which have been diversified and to add to its feathers more recently the inoculation delivery methods, diagnosis of diseases, and cosmetics in the commercial industry are gaining more application based interests.A very primitive application of these MNs was employed in drug transportation with the use of solid-based silicon MN . A dissoIn most cases, a dissolvable type of MN is considered as the best viable option for delivering the vaccine. In the past few years, the conventional hypodermic needles were commonly used for administering the vaccine, which is getting replaced with the dissolving type of MNs, which exhibit decent biocompatibility, appear to be robust with scalable properties, and are eco-friendly since they tend to cause the least amount of bio hazardous wastage . To theiDiagnosing diseases and determining the effectiveness of therapies can also be achieved by looking into the biomarkers which are present in the body for evaluation of health. The existing procedures tend to be painful for the patient undergoing the test and require some personalized equipment . NeverthMicroneedles are thought to be an effective cosmetic treatment for aging, skin lesions, vulgaris, and wrinkles . Kim et This paper depicts polymeric microneedles as a cutting-edge technology in the biomedical and pharmaceutical fields and presents a diversified scope for the future science and research areas. There has been remarkable and progressive research in transdermal routes of drug delivery through polymeric microneedles in the previous decade. This comprehensive review sums up the MNs technologies in the transdermal drug delivery era with regard to polymers and their types of materials, drug transportation kinetics, insertion mechanisms with mathematical modeling of polymeric MNs, and advances in fabrication methods, which are explored vividly. In the last sections, this paper even depicts the underlying gaps and the existing challenges which need to be met in MN research to realize an efficient means of low-cost fabrication. Finally, the paper wraps up with polymeric MN applications in diversified areas.


In the absence of catenin signaling, phrenic MN topography is eroded, MN clustering is lost and phrenic axons and dendrites fail to grow appropriately. Despite the essential requirement for catenins in early phrenic MN development, they appear to be dispensable for phrenic MN maintenance, as catenin deletion from postmitotic MNs does not impact phrenic MN topography or function. Our data reveal a fundamental role for catenins in PMC development and suggest that distinct mechanisms are likely to control PMC maintenance.Phrenic Motor Column (PMC) neurons are a specialized subset of motor neurons (MNs) that provide the only motor innervation to the diaphragm muscle and are therefore essential for survival. Despite their critical role, the mechanisms that control phrenic MN development and function are not well understood. Here, we show that catenin-mediated cadherin adhesive function is required for multiple aspects of phrenic MN development. Deletion of β- and γ- Breathing is an essential motor behavior that is required for survival. In mammals, contraction of the diaphragm muscle is critical for bringing oxygenated air into the lungs during inspiration . DiaphraWe previously identified a distinct combinatorial cadherin code that defines phrenic MNs, which includes both the broadly expressed type I N-cadherin and a subset of specific type II cadherins , 2022. Ccatenin, mice display severe respiratory insufficiency, gasp for breath, and die within hours of birth. Using phrenic nerve recordings, we determined that catenins are crucial for respiratory motor output, as MN-specific catenin inactivation leads to a striking decrease in phrenic MN activity. We further show that catenins are required to establish phrenic MN cell body settling position, as well as PMC axonal and dendritic morphology. Finally, we show that catenins are only required for PMC development and function during a narrow developmental window, as catenin deletion from postmitotic MNs does not impact respiratory output. Our data demonstrate a fundamental role for the cadherin-catenin cell adhesion complex in phrenic MN development and respiratory function and indicate that distinct pathways likely act to maintain PMC function.Here, we show that catenin activity is required for proper respiratory behavior and robust respiratory output. After MN-specific deletion of β- and γ-catenin , mouse anti-olig2-A488-conjugated , rabbit anti-β-Catenin , goat anti-Scip , mouse anti-Islet1/2  (RRID:AB_887743), rabbit anti-synaptophysin , and α-bungarotoxin Alexa Fluor 555 conjugate . Images were obtained with a Zeiss LSM 800 confocal microscope and analyzed with Zen Blue, ImageJ (Fiji), and Imaris (Bitplane). Phrenic MN number was quantified using the Imaris “spots” function to detect cell bodies that coexpressed high levels of Scip and Isl1/2 in a region of interest limited to the left and right sides of the ventral spinal cord.escribed , on tissescribed . The fol2314683) , rabbit For labeling of phrenic MNs, crystals of carbocyanine dye, DiI  were pressed onto the phrenic nerves of eviscerated embryos at e18.5, and the embryos were incubated in 4% PFA at 37°C in the dark for 4–5 weeks. Spinal cords were then dissected, embedded in 4% low melting point agarose (Invitrogen) and sectioned using a Leica VT1000S vibratome at 100–150 μm.x and y coordinates. Coordinates were expressed relative to the midpoint of the spinal cord midline, defined as position x = 0, y = 0. To account for experimental variations in spinal cord size, orientation, and shape, sections were normalized to a standardized spinal cord whose dimensions were empirically calculated at e13.5 . We analyzed every other section containing the entire PMC .MN positional analysis and correlation analysis were performed as previously described , 2019. MFor the analysis of dendritic orientation, we superimposed a radial grid divided into eighths (45° per octant) centered over phrenic MN cell bodies spanning the entire length of the dendrites. We drew a circle around the cell bodies and deleted the fluorescence associated with them. Fiji (ImageJ) was used to calculate the fluorescent intensity (IntDen) in each octant which was divided by the sum of the total fluorescent intensity to calculate the percentage of dendritic intensity in each area.3, 0.5 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2, and 30 mM D-glucose and was equilibrated by bubbling in 95% O2/5% CO2. The hindbrain and spinal cord were exposed by ventral laminectomy, and phrenic nerves exposed and dissected free of connective tissue. A transection at the pontomedullary boundary rostral to the anterior inferior cerebellar artery was used to initiate fictive inspiration. Electrophysiology was performed under continuous perfusion of oxygenated Ringer’s solution from rostral to caudal. Suction electrodes were attached to phrenic nerves just proximal to their arrival at the diaphragm. The signal was band-pass filtered from 10 Hz to 3 kHz using AM-Systems amplifiers (Model 3000), amplified 5,000-fold, and sampled at a rate of 50 kHz with a Digidata 1440A (Molecular Devices). Data were recorded using AxoScope software (Molecular Devices) and analyzed in Spike2 (Cambridge Electronic Design). Burst duration and burst activity were computed from 4 to 5 bursts per mouse, while burst frequency was determined from 10 or more minutes of recording time per mouse. Burst activity was computed by rectifying and integrating the traces with an integration time equal to 2 s, long enough to encompass the entire burst. The maximum amplitude of the rectified and integrated signal was then measured and reported as the total burst activity.Electrophysiology was performed as previously described . Mice weConscious, unrestrained P0 mice were placed in a whole body, flow-through plethysmograph (emka) attached to a differential pressure transducer (emka). We modified 10 ml syringes to use as chambers, as smaller chambers increase signal detection in younger mice. Experiments were done in room air . Mice were placed in the chamber for 30 s at a time, for a total of three to five times, and breathing parameters were recorded. Mice were directly observed to identify resting breaths. At least ten resting breaths were analyzed from every mouse. Data are presented as fold control, where the control is the average of 2 littermates in normal air.p-values were calculated using unpaired, two-tailed Student’s t-test. Burst activity data in p-values were calculated using the Mann–Whitney test. p < 0.05 was considered to be statistically significant, where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Data are presented as box and whisker plots with each dot representing data from one mouse unless otherwise stated. Small open squares in box and whisker plots represent the mean.For all experiments a minimum of three embryos per genotype, both male and female, were used for all reported results unless otherwise stated. The Shapiro–Wilk test was used to determine normality. All data, with the exception of electrophysiology burst activity data in N-cadherin (N-cad) and the type II cadherins Cdh6, 9, 10, 11, and 22 . β- and γ-catenin are obligate intracellular factors required for cadherin-mediated cell adhesive function and catenin deletion enables us to interrogate the full repertoire of cadherin actions in phrenic MNs  by e11.5 0 , while respiratory frequency is not affected 18.5/P0, shortly before βγ-catMNΔ mice die. We observed a striking reduction in the activation of phrenic MNs in βγ-catMNΔ mice (catMNΔ mice were either of very low amplitude (∼85%) or non-detectable (∼15%). After rectifying and integrating the traces, we found a nearly 70% decrease in total burst activity in βγ-catMNΔ mice (In order to assess breathing in βγ-day (P)0 . We founaffected . This reaffected , indicatarations . These pMNΔ mice . While bMNΔ mice . Our datcatMNΔ mice? We asked whether early phrenic MN specification, migration and survival are impacted after catenin inactivation. We acquired transverse spinal cord sections through the entire PMC at e11.5 and stained for the TF Olig2 to label MN progenitors and the phrenic-specific TF Scip and MN-specific TF Isl1/2 to label all phrenic MNs . Bγ-catMNΔ mice displayed a significant shift in phrenic MN cell body position, with cell bodies shifting ventrally toward the edge of the spinal cord and toward the midline , representing 40–45% of the overall dendritic intensity . VentralcatMNΔ mice at e18.5. We found that βγ-catMNΔ mice display a lack of innervation in the ventral diaphragm . βγ-catChATMNΔ mice show MN-specific loss of catenin expression by e13.5 , we do not see a loss of clustering in these mice .The original contributions presented in this study are included in the article/This animal study was reviewed and approved by the Institutional Animal Care Use Committee of Case Western Reserve University .AV and PP conceived the project and wrote the manuscript. AV, MM, RL, AA, LL, and PP performed the experiments and analyzed the data. NZ provided the reagents. All authors contributed to the article and approved the submitted version.


The results clearly demonstrate that item recall is poorer for similar lists than dissimilar lists when similarity is defined in this way.The phonological similarity effect in short‐term memory (STM) is the finding that serial recall of lists of similar sounding items is poorer than that of dissimilar sounding items. This is traditionally argued to be due to a detrimental effect on memory for the order of the words in the list and not on memory for the words themselves. Models that propose forgetting from STM is due to interference must invoke an additional compensatory process where the shared element of the words acts as a cue to recall, in order to account for the lack of an effect on memory for the words. However, the possibility of a detrimental effect of phonological similarity on item memory when these compensatory processes are not available has not been investigated. Two experiments ( The phonological similarity effect, poorer serial recall of lists of words that sound similar to each other than of lists of dissimilar words, is a cornerstone of the literature on short‐term memory (STM) that has shaped theories of memory for over half a century. First described by Conrad and Hull  by compaShort‐term memory is an area where there are numerous computational models that attempt to simulate human memory performance, and models can be pitted against each other to determine which provides the closest approximation to human data  words as stimuli in serial recall, which are summarized in Table One very common operational definition of phonological similarity has been to make all the stimuli in the list rhyme. Before describing the research on CVC words, it is worth noting that the view that phonological similarity only affects the order of recall may have arisen because many of the early studies used letters as stimuli, and this practice is still common. Letters afford the selection of a familiar set of stimuli that rhyme, and a set that are relatively dissimilar, but this means they are necessarily drawn from a small set with repeated presentation of stimuli across lists. This methodology is likely to reduce item errors in recall and maximize order errors.Focussing on those studies that have used CVC word stimuli, Table As can be seen from Table When the rhyme was unique to each list and the results were scored by a strict in‐position criterion, Nimmo and Roodenrys . Unfortunately, his data were reported only for the number of entire sequences recalled correctly, so do not provide evidence regarding the impact on recall of items without respect to position. However, Coltheart  which may have created a level of confusion between items across similar lists, which was not present for the dissimilar lists. Consistent with this alternative explanation, Coltheart's results also revealed that half of the difference in recall of the similar and dissimilar items could be accounted for by explicit intrusions of items from the previous lists. It is worth noting that Fallon et al.  function as a cue. It is even possible that a cue may impair recall in comparison with a dissimilar list if the cue is actually inefficient and its use taxes the system. It is often stated that words are more readily cued by their beginning than other components. Nelson and Garland  demonstrIn addition, many studies have used dissimilar conditions that allowed for some words in a list to share phonemes, which might be expected to reduce the difference in item recall if interference does occur. What appears to be critical in assessing the claim that similarity does not affect item recall in STM is observing a detrimental effect in the absence of other possible explanatory factors. From the review above, only Experiment 3 of Nimmo and Roodenrys  appears tap, hug, gym, boss, cord and rail). Nimmo and Roodenrys . In the other two conditions, each word shares two of its three phonemes with another word in the list. Each possible pair is shared in two of the six words in a list. The two conditions differ in how they are shared. In the similar consistent condition, the two phonemes of a word are shared with another word, so two words in the list rhyme, two share the two consonants, and two share the initial consonant and vowel . In the similar inconsistent condition, the two phonemes of each word are shared with two different words in the list so no word shares more than a single phoneme with any other word . Therefore, the potential for a phoneme to be lost from a word, all other things being equal, is the same across the two similar conditions, but the impact is expected to be different. In the consistent condition, the loss of one of the three phonemes from a word will make it confusable with one other word in the list. For instance, in the example provided above, the loss of the first phoneme /l/ of the word leaf, but not the loss of the phoneme /i/ or /f/ will make it indistinguishable from reef. However, this is not the case for the inconsistent condition where any two phonemes of the word are sufficient to discriminate it from the other words in the list. For instance, in the example above, after losing any phoneme of the word cap, it will still be possible to distinguish it from all other list items.The experiments reported below aim to provide a test of the assertion that phonological similarity influences order memory but not item memory. More specifically, the two experiments involve four conditions, the second experiment being a replication of the first with a second set of stimuli. The first condition is the control, dissimilar one. In the first experiment, some lists have some phonemes occurring twice in a list in this condition but in different intrasyllabic positions, whereas in the second experiment no phoneme was repeated within a six‐item list , and 35 were male. The study took approximately 10 min to complete, and participants were paid £1.50. The data from one participant were lost due to a problem with the computer system.Sixty‐one adult participants from the Prolific participant recruitment platform took part in the experiment. They were all native English speakers from North America, Britain or Australia with an approval rating of at least 90% on Prolific. They had a mean age of 33.7 years (f = 1.20) reported in Experiment 3 of Nimmo and Roodenrys (f = 0.24).Based on the effect size  = 172.58, p < .001, ηp2 = 0.82. Tukey's HSD tests revealed the words in the rhyming lists were more similar to each other  than the consistent and inconsistent conditions , which did not differ from each other, but were more similar than the dissimilar condition .All the stimuli used were CVC words. Five lists of six words were created for each condition, as described above (see Appendix A). The words in each set were matched on several lexicosemantic dimensions . Homophones of the presented word or misspellings that preserved the phonology were also scored as correct . In total, 1.3% of items were corrected. Over half of these were in the rhyming condition where the rhyming words appear to have primed the incorrect spelling.Data are available in the Open Science Framework repository, M = 0.56, SD = 0.29) compared with the other conditions. The performance of the participants was similar across the rhyming condition , the consistent condition  and the inconsistent condition . The analysis of variance confirmed those trends. The analysis of variance revealed a main effect of condition, F = 11.91, p < .001, ηp2 = 0.17, a main effect of serial position, F = 216.87, p < .001, ηp2 = 0.79, and a two‐way interaction between those factors, F = 2.22, p = .005, ηp2 = 0.04. Post‐hoc Tukey's HSD tests revealed the participants performed better in the dissimilar condition compared with all other conditions, all ps < 0.001, all Cohen's d > 0.529, which did not differ one from another, all ps > 0.423, all Cohen's d < 0.202. The interaction simply reflects the growing impact of phonological similarity as one moves towards the end of the list.Overall, recall performance of the participants when assessed via strict scoring was superior in the phonologically dissimilar condition  compared with the other conditions. Participants were also better in the dissimilar condition  relative to the consistent condition , which was superior to the inconsistent condition . The analysis of variance confirmed these trends. Again, the analysis of variance revealed a main effect of condition, F = 33.65, p < .001, ηp2 = 0.36, a main effect of serial position, F = 93.71, p < .001, ηp2 = 0.61, and a two‐way interaction between those factors, F = 2.76, p < .001, ηp2 = 0.05. Post‐hoc Tukey's HSD tests revealed the participants performed better in the rhyming condition compared with all other conditions, all ps < 0.003, all Cohen's d > 0.398. Post‐hoc analysis also revealed that participants performed better in the dissimilar condition compared with the consistent condition, p = .033, Cohen's d = 0.330 and that participants performed better in the consistent condition compared with the inconsistent condition, p = .002, Cohen's d = 0.439.Participants' responses were assessed via lenient scoring, revealing that performance was superior when the to‐be‐remembered items were rhyming  than the other conditions. This examination also revealed that the proportion of order errors was larger in the rhyming condition  while the proportion of order errors was similar between the consistent condition  and the inconsistent condition . The analysis of variance confirmed the presence of a main effect of condition, F = 31.28, p < .001, ηp2 = 0.35. Post‐hoc Tukey's HSD tests revealed that the proportion of order errors was larger in the rhyming condition compared with the other conditions, all ps < 0.001, all Cohen's d > 0.816. The proportion of order errors in the dissimilar condition was smaller than the proportion of order errors in the rhyming condition , and in the consistent condition , but not the inconsistent condition . Then, post‐hoc analysis confirmed that the proportion of order errors was similar between the consistent condition and the inconsistent condition .Examination of conditional order errors revealed that the proportion of order errors was lower in the dissimilar condition  = 9.20, p < .001, ηp2 = 0.135. Post‐hoc Tukey's HSD tests were conducted on all comparisons but, for the sake of brevity, only significant differences will be reported. There were significantly more omissions in the consistent and inconsistent conditions than the dissimilar condition , or the rhyming condition .The proportion of responses made up of omissions, intrusions and repetitions of a list word during recall are shown in Table F = 36.97, p < .001, ηp2 = 0.385. Post‐hoc Tukey's HSD tests revealed that there were fewer intrusions in the rhyming condition than the other conditions . There were also significantly more intrusions in the inconsistent condition than the consistent or dissimilar conditions .There was a significant effect of condition in the analysis of the intrusion proportions, F = 11.69, p < .001, ηp2 = 0.165. Post‐hoc Tukey's HSD tests revealed that there were more repetitions in the rhyming condition than the other conditions .There was a significant effect of condition in the analysis of the repetition errors, The results of the first experiment clearly demonstrate the standard finding of better recall of items in their correct position when they were phonologically dissimilar to each other in comparison with any of the similar conditions. They also replicate the finding that recall of the items without respect to serial position is superior for rhyming lists over dissimilar lists , and 42 were female. As mentioned above, 60 participants were planned, but due to technical difficulties, 57 participants composed the final sample.Fifty‐seven undergraduate Psychology students from an Australian University participated as part of a course requirement. The average age was 20.3 years  = 319.23, p < .001, ηp2 = 0.85. Tukey's HSD tests revealed the words in the rhyming lists were more similar to each other  than the consistent and inconsistent conditions , which did not differ from each other, but were more similar than the dissimilar condition .As in Experiment 1, all the stimuli used were CVC words. Seven lists of six words were created for each condition (see Appendix B). The sets of words were again matched on several lexicosemantic dimensions . Like Experiment 1, participants' performance was of comparable level across the rhyming condition , the consistent condition  and the inconsistent condition . The analysis of variance revealed a main effect of condition, F = 9.30, p < .001, ηp2 = 0.14, a main effect of serial position, F = 211.85, p < .001, ηp2 = 0.79 and a two‐way interaction between those factors, F = 2.44, p = .002, ηp2 = 0.04. Post‐hoc Tukey's HSD tests confirmed the descriptive trends. More exactly, the analysis revealed that participants' performance was superior in the dissimilar condition compared with all other conditions, all ps < 0.001, all Cohen's d > 0.426, which did not differ one from another, all ps > 0.575, all Cohen's < 0.209. Once again, the difference across conditions was larger for later than initial serial positions.When participants' responses were assessed via strict scoring, the performance of the participants was superior in the dissimilar condition  compared with the dissimilar condition , which was superior to both the consistent  and the inconsistent conditions . Once again, the analysis of variance confirmed those trends. Like Experiment 1, there was a main effect of condition, F = 69.54, p < .001, ηp2 = 0.55, a main effect of serial position, F = 93.57, p < .001, ηp2 = 0.63, and a two‐way interaction between those factors, F = 4.69, p < .001, ηp2 = 0.08. Post‐hoc Tukey's HSD tests confirmed that all conditions differ one from another, all ps < 0.001, all Cohen's d > 0.503, except the inconsistent similar condition and the consistent similar condition which did not differ one from another, p = .212, Cohen's d = 0.312.Overall, with the lenient scoring, the performance was superior in the rhyming condition  was again inferior to the other conditions. Echoing the results of Experiment 1, there were more order errors in the rhyming condition  compared with the consistent condition  and the inconsistent condition . The analysis of variance confirmed the presence of a main effect of condition, F = 39.48, p < .001, ηp2 = 0.41. Post‐hoc Tukey's HSD tests revealed that the proportion of order errors differ one from another in all four conditions, all ps < 0.038, all Cohen's d > 0.446, except the inconsistent condition and the consistent condition, p = .831, Cohen's d = 0.129.Exploration of conditional order errors revealed that the proportion of order errors in the dissimilar condition  = 21.876, p < .001, ηp2 = 0.281. Post‐hoc Tukey's HSD tests were conducted on all comparisons but, for the sake of brevity, only significant differences will be reported. There were significantly fewer omissions in the rhyming condition than the other conditions . There were also significantly fewer omissions in the dissimilar condition than the inconsistent condition .The proportion of responses made up of omissions, intrusions and repetitions of a list word during recall are shown in Table F = 36.48, p < .001, ηp2 = 0.394. Post‐hoc Tukey's HSD tests revealed that there were fewer intrusions in the rhyming condition than the other conditions . There were also significantly more intrusions in the inconsistent condition than the dissimilar condition .There was a significant effect of condition in the analysis of the intrusion proportions, F = 17.11, p < .001, ηp2 = 0.234. Post‐hoc Tukey's HSD tests revealed that there were more repetitions in the rhyming condition than the other conditions . There were also significantly more repetitions in the consistent condition than the dissimilar condition .There was a significant effect of condition in the analysis of the repetition errors, The results of the second experiment are extremely similar to those of the first experiment, confirming the soundness of our findings. On the strict, in‐position scoring, performance was better in the dissimilar condition than the similar conditions, which did not differ from each other. On the lenient scoring, recall of the items was again significantly better if the list of words rhymed than if they were dissimilar and, again, significantly worse than the dissimilar condition in the other similarity conditions. The one difference of note is that in this experiment the level of item recall was equivalent between the similar consistent and similar inconsistent conditions.The results of these two experiments clearly demonstrate that recall of the words in the list, independent of order, is impaired by phonological similarity when similarity is not defined by the rhyme. Some previous studies have also demonstrated this effect, but the current experiments extend on those by demonstrating that this is the case when there was no component common to all the items in a list that might act as a cue to recall the items, and free of proactive interference effects from having the same words presented in previous trials. In the similar conditions, two phonemes from each word were present in other words in the list, and in both experiments, these conditions were recalled significantly less well than the dissimilar condition. Contrary to the claim that similarity only affects the order of recall, the magnitude of the difference observed between these conditions in the lenient scoring is only slightly smaller than the difference observed in the strict scoring, suggesting much of the effect in the strictly scored data is due to the loss of information about the identity of the items, rather than an effect on order of recall.The two similar conditions differ in how the phonemes were shared across the words in a list. In the consistent condition, the two phonemes of a word that were shared, were shared with one other word, so two words in the list rhymed, two shared the onset and vowel, and two shared the two consonants. In the inconsistent condition, the two phonemes of a word that were shared, were shared with different words so no two words overlapped on more than a single phoneme.pad and patch, or the rhyme, might occur in sequence and if the participant notices, it may be an effective cue to recall the second word , whereas in Experiment 2 they did not differ significantly (0.63 vs. 0.60), although the difference was in the same direction. In both experiments, the lower item recall in the inconsistent condition was mirrored by an increase in intrusions. It is possible that a small difference in recall, and intrusions, arises because on some lists the common component may assist in recall in a redintegration process after the degraded representations have been retrieved (Schweickert, Computational models of STM have modelled the effect of phonological similarity as impacting the order of recall, but not necessarily the probability of recalling an item anywhere in the list (e.g., Lewandowsky & Farrell, This paper is the first to have deliberately sought to challenge the widely accepted notion that phonological similarity affects only the order of recall and not the likelihood of recalling an item in serial recall. It demonstrates that items which share phonemes are less well recalled, irrespective of serial position, and therefore offers a new constraint on theories of STM.Steven Roodenrys: Conceptualization; formal analysis; investigation; methodology; project administration; writing – original draft; writing – review and editing. Dominic Guitard: Conceptualization; data curation; formal analysis; investigation; methodology; visualization; writing – original draft; writing – review and editing. Leonie M. Miller: Conceptualization; methodology; writing – original draft; writing – review and editing. Jean Saint‐Aubin: Conceptualization; methodology; writing – original draft; writing – review and editing. Jeffrey M. Barron: Investigation; software; writing – review and editing.All authors declare no conflict of interest.


Taking MG63 osteoblasts as the research object, they were inoculated on glass slides (G group), polished titanium sheets (P group), and sandblasted acid-base treated pure titanium sheets (S group). In addition, FSS of 0 dunes/cm2 (static group) and 12 dunes/cm2 (stress group) were given, respectively. Then, quantitative reverse transcription-PCR (RT-qPCR) and western blot were used to detect the mRNA and protein expressions of low-density lipoprotein receptor-related protein 5 (LRP5) and β-catenin in MG63 cells. The results showed that the expression levels of β-catenin mRNA and protein in cells in the stress group were significantly increased (P < 0.05), and the protein expression level of LRP5 was significantly decreased (P < 0.05). The expression level of LRP5 in group S was greatly inhibited, while the expression level of β-catenin was significantly upregulated. Therefore, FSS can stimulate the expression of LRP5 and β-catenin in osteoblasts. Fluid shear stress can promote osteoblast proliferation in vitro; the Wnt/β-catenin signaling pathway is involved in regulating fluid shear stress to promote osteoblast proliferation.In order to investigate the effect of fluid shear stress on the proliferation of osteoblasts and the regulatory role of the Wnt/ Implant denture has gradually become an important trend in the repair of dentition defects and loss, and the success rate of clinical application is high, but there were some individual failure cases . The sucAmong all the stresses, mechanical tension and flow shear stress (FSS) are the most important forces on the implant-bone interface . There iTherefore, the expression changes of Wnt/beta-catenin scanning signaling pathway-related mRNA and protein in MG63 cells after FSS treatment were analyzed. The purpose of this study was to investigate the effects of the Wnt signaling pathway and biomechanics on osteoblast activation and to provide more evidence for biomechanical studies of implant-bone interface cells.β-catenin antibody was purchased from a cell signaling technology company, USA. Rabbit antihuman β-actin antibody and rabbit antihuman α-tubulin antibody were purchased from Shanghai Beyotime Biotechnology Co., Ltd.MG63 cells were purchased from ATCC, USA. TA0 grade Shengye pure titanium plate was purchased from Shaanxi Baoji three-line Nonferrous Metal material factory. BCIP/NBT alkaline phosphatase color reagent kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. Alizarin Red was purchased from Shanghai Beyotime Biotechnology Co., Ltd. Trizol reagent total RNA extraction kit was purchased from Invitrogen, USA. The RNA reverse transcription kit was purchased from Invitrogen, USA. SYBR Green I detection kit was purchased from Invitrogen, USA. Dulbecco's modified Eagle's medium (DMEM) low-sugar medium was purchased from Hangzhou Genom Company. Fetal bovine serum was purchased from Gibco, USA. Penicillin and streptomycin were purchased from Hyclone, USA. The nuclear protein extraction kit was purchased from Merck, Germany. Rabbit antihuman LRP5 antibody was purchased from a cell signaling technology company, USA. Rabbit antihuman 2 and 100% humidity. The cell culture medium was replaced every 48 h. When the confluence of cells reached about 80%, the cells were digested with 0.25% trypsin, and the cells were subcultured. After a 3–4 d culture, FSS treatment was performed.MG63 osteoblasts were cultured in a DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, respectively and placed in a 37°C incubator containing 5% COThe rectangular specimen with a length of 75 mm, a width of 25 mm, and a thickness of 1 mm was processed by pure titanium plate wire cutting. Smooth slides of the same size were used as the control group, which were rolled into polishing treatment and sandblasting-acid and alkali treatment according to the processing methods. They were named group G, group P, and group S, respectively, and their surface morphology was observed by scanning electron microscopy.2. After being pumped from the peristaltic pump, the perfusion fluid enters the flow cavity through the pipeline, applies corresponding shear stress to the cells, and then enters the liquid storage bottle through the pipeline, thereby forming a closed circulation system.The flow chamber system designed by Hochmuch et al.  was refe2 (stationary group) and 12 dynes/cm2 (stressed group). Primary osteoblasts and MG63 osteoblasts were collected at 0 h, 0.25 h, 0.5 h, 1 h, 2 h, and 4 h after stress. Trizol reagent was used to extract total RNA from cells, and the purity and concentration were detected. cDNA reverse transcription was carried out according to the cDNA reverse transcription kit, and the products were stored at −20°C for later use. Primers for quantitative detection of LRP5 and β-catenin mRNA sequences were designed and synthesized, and the specific information is shown in Cells after stress were rolled into 0 dynes/cmβ-catenin , rabbit antihuman β-actin , and rabbit antihuman α-tubulin  were added and incubated overnight at 4°C. The primary antibody was recovered and washed with Tris-buffered saline/Tween (TBST) three times. The diluted secondary antibody was added and incubated at room temperature for 2 h. The membrane was washed with TBST three times. The ECL protein development kit was used to develop the protein bands of the sample, and the gel imaging system  was used for image acquisition and image analysis. The data were standardized with the household-looking genes β-actin and α-tubulin as an internal reference for LRP5 and β-catenin, respectively. All experiments were repeated three times.MG63 osteoblasts were collected and lysed at 0 h, 0.25 h, 0.5 h, 1 h, 2 h, and 4 h after stress and total protein was extracted. The standard curve of the extracted protein concentration was drawn using the BCA protein concentration detection kit, so as to carry out the quantitative detection of the sample protein. 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) adhesive was configured, and electrophoretic separation of proteins was performed after sample loading. At the end of electrophoresis, the PVDF membrane was transferred with proteins and then sealed with the sealing solution of skimmed milk powder. The sealing condition was 80 rpm at room temperature for 2–4 h. Diluted rabbit antihuman LRP , rabbit antihuman P < 0.05 was considered to be statistically considerable.SPSS 19.0 was used for statistical analysis of experimental data, and all experimental data were expressed as mean ± standard deviation. Differences between groups were statistically analyzed by one-way analysis of variance, and The culture identification of the newly inoculated cells was performed to observe the adherent growth state of the cells during the culture process. In Morphological differences in titanium sheets after different surface treatments were observed by scanning electron microscopy (SEM), as presented in P > 0.05). However, the overall expression level of LRP5 mRNA in group G was considerably higher than that in group S (P < 0.01). The expression level of LRP5 mRNA in each group was the highest at 1 h, and the expression level of LRP5 mRNA in each group was the lowest at 0 h.The differences in LRP5 mRNA levels in MG63 cells on different titanium planting surfaces after FSS treatment were detected, and the results were shown in β-catenin mRNA levels in MG63 cells on different titanium planting surfaces after FSS treatment was detected, and the results are shown in β-catenin mRNA in MG63 cells in the stressed group was considerably higher than that in the stationary group (P < 0.01). There was no remarkable difference in β-catenin mRNA levels in the stationary group at different time points (P > 0.05). Except for those at 0 h and 1 h, the expression level of group G was always the highest at other time points. Except for those at 2 h and 4 h, the expression level of group S was always the lowest at other time points. The levels of β-catenin mRNA in the stressed group were considerably different at different time points (P < 0.01). After FSS treatment for four hours, the levels of β-catenin mRNA in group P and group S were considerably higher than those in group G (P < 0.01).The difference in P < 0.01). Under different FSS treatments, the LRP5 protein expression level of group G was considerably higher than that of group P and group S (P < 0.05), but there was no remarkable difference between the expressions of group S and group P (P > 0.05). The expression levels of each group were high at 0.25 h, 0.5 h, and 1 h. After 4 h of treatment, the expression level of LRP protein in group G was considerably higher than that in group S and group P (P < 0.01). The expression level of LRP protein in group S in the stressed group was considerably higher than that in group G and group P (P < 0.01).The changes in the expression level of LRP5 protein in MG63 cells on different titanium planting surfaces after FSS treatment were detected, and the results are shown in β-catenin protein expression in MG63 cells on different titanium seeding surfaces after FSS treatment were analyzed, and the results are shown in β-catenin protein expression level in MG63 cells of the stressed group and the stationary group suggested that the average β-catenin protein expression level in the MG63 cells of the stressed group was considerably higher than that in the stationary group (P < 0.01). The expression level of group S was considerably higher than that of group G and group P (P < 0.05), but there was no remarkable difference between the expressions of group G and group P (P > 0.05). The expression level of β-catenin protein in both the stressed group and the stationary group was the lowest at 0 h. Moreover, the expression level of β-catenin protein in the three groups of titanium-planted surface MG63 cells under different FSS showed a trend of first increasing and then decreasing over time.The changes in 2+ pathway, and Wnt/JNK/Planar cell polarity pathway (PCP pathway) [β-catenin [β-Catenin is a core member of the Wnt/beta-catenin scanning signaling pathway. When the Wnt signaling pathway is activated, Wnt can interact with Frizzled and LRP5 receptor, and then activate LRP5 and induce axin binding, activate Frizzled and induce Dsh binding. Dsh protein activates the phosphorylation of GSK3β and ultimately inhibits the phosphorylation of β-catenin [At present, many studies showed that giving corresponding biomechanical stimulation can promote cell proliferation, activation-related cell signaling pathways, and activates the expression of a series of target genes –16. The pathway) , 18. The-catenin . As an i-catenin , 21. β-Cβ-catenin [β-catenin can activate the expression of downstream target genes that regulate the cell cycle, and thus participate in the proliferation pathway of chondrocytes. Exploration of the effect of FSS stimulation on the expression levels of LRP5 and β-catenin mRNA and protein in cells showed that the FSS of 12 dynes/cm2 can promote the expression level of β-catenin mRNA and protein without affecting the expression level of LRP5 mRNA but can inhibit the expression level of LRP5 protein. Studies pointed out that the expression of LRP5 mRNA and protein in MG63 cells on the titanium surface was inhibited after sandblasting and acid-base treatment and the expression of β-catenin was promoted. The surface properties of titanium after sandblasting and acid-base treatment can promote the proliferation and differentiation of MG63 cells, and this effect can be achieved by activating the Wnt/beta-catenin scanning signaling pathway. Wang et al. researched and analyzed the surface and smoothness of nanostructured titanium, and it was revealed that the apoptosis rate of upper MG63 cells was low, and there was no remarkable difference between the groups. However, the β-catenin pathway was obviously activated in the cells on the surface of nanostructured titanium and the osteogenic activity was enhanced [β-catenin in the Wnt/beta-catenin scanning signaling pathway after FSS and sandblasting acid-base treatment.Bone tissue often adapts to changes in mechanical loading through changes in structure and density. In this process, bone cells are the first to perceive changes in bone tissue . Bone ce-catenin . It has -catenin  confirme-catenin  showed tenhanced . In summ2 FSS and sandblasting acid-base treatment on the titanium surface can promote the expression of β-catenin and inhibit the expression of LRP5, which in turn plays a role in promoting osteoblast proliferation. However, this work only explores the effect of FSS on cell proliferation in vitro and the expression levels of LRP5 and β-catenin in the Wnt/beta-catenin scanning signaling pathway. The proliferation activity and apoptosis rate of osteoblasts after different surface treatments were not detected in this study. In the follow-up, it is necessary to further establish corresponding animal models to verify the regulatory mechanism in vivo, and the Wnt/beta - catenin scanning pathway inhibitors shall be added to explore the effect of this pathway on the proliferation and differentiation level, osteogenesis ability of osteoblasts. In conclusion, this study initially explores the mechanism of cellular mechanics signal transduction related to the implant-bone interface, which lays the foundation for further research on the cell biomechanics of the implant cell interface.In summary, 12 dynes/cm


Col1a1, Alp, Runx2, Opn, and Ocn). Furthermore, dopamine inactivated AKT/GSK-3β/β-catenin signaling pathway. Treatment of LiCl (GSK-3β inhibitor) rescued the inhibitory effects of dopamine on osteogenic differentiation of rBMSCs. LY294002 (AKT inhibitor) administration exacerbated the inhibitory effects of dopamine on osteogenic differentiation of rBMSCs. Taken together, these findings indicate that dopamine suppresses osteogenic differentiation of rBMSCs via AKT/GSK-3β/β-catenin signaling pathway. Our study provides new insights into the role of neurotransmitters in bone homeostasis.Nervous system is critically involved in bone homeostasis and osteogenesis. Dopamine, a pivotal neurotransmitter, plays a crucial role in sympathetic regulation, hormone secretion, immune activation, and blood pressure regulation. However, the role of dopamine on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (rBMSCs) remains poorly understood. In this study, we firstly investigated the effect of dopamine on the apoptosis, proliferation, and osteogenic differentiation of rBMSCs. Dopamine did not, however, interfere with the apoptosis and proliferation of rBMSCs. Interestingly, dopamine suppressed the osteogenic differentiation of rBMSCs, as characterized by reduced ALP staining, ALP activity, mineralized nodule formation, and the mRNA and protein levels of osteogenesis-related genes ( Osteoporosis is a systemic skeletal disease characterized by a reduction in bone mineral density and deterioration of bone structure, leading to an increased risk of bone fragility and fractures . OsteopoBone remodeling is a dynamic, physiological, and continuous process coupling osteoblastic bone formation and osteoclastic bone resorption . This prDopamine (DA), a pivotal catecholamine neurotransmitter of the central nervous system, plays crucial roles in cognition, emotion, memory, attention, vision, reward, sympathetic regulation, and hormonal regulation . Abnormaβ-catenin signaling pathway is considered the master regulator involved in osteogenesis [β-catenin, induces nucleus translocation of accumulated β-catenin, and thus promotes osteogenic differentiation. β-Catenin can be regulated by glycogen synthase kinase 3β (GSK-3β), which acts as a negative regulator of Wnt/β-catenin signaling pathway and is implicated in governing osteogenic differentiation. In addition, activation of AKT -AKT) can trigger GSK-3β phosphorylation at Ser9, which leads to the inactivation of GSK-3β, and then inhibit the degradation of β-catenin [Currently, Wnt/ogenesis . Wnt sig-catenin –21.β/β-catenin signaling pathway in DA-induced osteogenic differentiation remains to be elucidated. Thus, in this study, firstly we isolated and identified rat bone mesenchymal stem cells (rBMSCs). Then, we evaluated the effect of DA on the apoptosis and proliferation of rBMSCs. Next, we carried out ALP staining, ALP activity assay, Alizarin Red staining, and mRNA and protein levels of genes related to osteogenic differentiation to investigate the effect of DA on osteogenic differentiation. Finally, we used GSK-3β inhibitor lithium chloride (LiCl) and AKT inhibitor LY294002 to explore the mechanism by which DA regulated osteogenesis. This study may provide new insights into the mechanisms underlying bone remodeling and a theoretical basis for the treatment of bone defects or osteoporosis.However, to the best of our knowledge, the role of AKT/GSK-3α-minimum essential medium  and centrifuged at 1000 rpm for 5 min. The cells were resuspended in growth medium containing 10% fetal bovine serum  and 1% Penicillin/Streptomycin  and cultured at 37°C in a 5% CO2 atmosphere. After 24 hours, the medium was replaced to remove the nonadherent cells and then was completely replaced every 2-3 days. Upon reaching 70–80% confluence, cells were trypsinized and passaged. Cells at passages 3-4 were used for subsequent experiments.The isolation and culture of rBMSCs from the tibias and femurs of 3-week-old male Sprague-Dawley rats were performed as described previously , 23. Briβ-glycerophosphate , 50 μg/ml ascorbic acid-2-phosphate , and 100 nM dexamethasone . Adipogenic or chondrogenic differentiation medium was purchased from Cyagen Biosciences. The expression of cell surface markers, including CD29, CD34, CD44, CD45, and CD90  on rBMSCs was identified by flow cytometry. Fluorescein isothiocyanate- (FITC-) conjugated mouse IgG  was used as a negative control.To investigate the multipotency of rBMSCs, the cells were induced to differentiate in osteogenic, adipogenic, or chondrogenic differentiation medium. For osteogenic differentiation, rBMSCs were cultured with osteogenic differentiation medium (OM), which is a growth medium supplemented with 10 mM 5 cells/well. After 24 h incubation, cultured cells were exposed to DA  at concentrations of 0, 10−9, 10−7, and 10−5 M for 2 days. Then, both floating and adherent cells were harvested and washed twice with precold PBS. The cells were centrifuged and resuspended in 100 μL of binding buffer containing 5 μL Annexin V-FITC and 10 μL PI. After incubation at room temperature in the dark for 15 min and adding 400 μL binding buffer, the mixture was analyzed by flow cytometry within 1 h.Apoptosis of rBMSCs was determined by using FITC-conjugated Annexin V and propidium iodide (PI) Apoptosis Detection kit  as per the manufacturer's instructions. Briefly, cells were seeded in 6-well plates at 103 cells/well. After 24 h incubation, cells were treated with dopamine at concentrations of 0, 10−9, 10−7, and 10−5 M for 1, 3, 5, and 7 days, respectively. The CCK-8 assays were performed using the CCK-8 Kit  according to the manufacturer's protocol.The toxicity of DA on rBMSCs was detected by the CCK-8 assay. Briefly, cells were seeded in 96-well plates at 3 × 10EdU assay was conducted by using EdU assay Kit  according to the manufacturer's instructions. In brief, cells were incubated in growth medium containing EdU for 2 h. Then, the cells were fixed in 4% formaldehyde  for 20 min and permeabilized with 0.3% TritonX-100  for 15 min. Subsequently, cells were incubated at room temperature for 30 min in the click reaction cocktail. Cell nuclei were stained by Hoechst 33342 for 30 min. Images were captured under confocal microscopy .After 7 days' osteogenic induction, ALP staining was performed according to the manufacturer's instructions . Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, rinsed twice with PBS, and then stained with ALP staining for 10 min. Images were captured under an inverted microscope .Cells were cultured in osteogenic differentiation medium for 7 days. For determination of ALP activity, cells were washed with PBS and assayed for ALP activity with an ALP Assay Kit  according to the manufacturer's recommendations. Total protein concentration was measured by using a BCA protein assay kit . The ALP activity was expressed as nmol phenol/15 min/mg protein.After the induction of osteogenic differentiation, mineral deposition was detected by Alizarin Red staining (ARS)  on day 14. Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, rinsed twice with PBS, and then stained with Alizarin Red staining for 5 min. Images were captured under an inverted microscope . For the quantification analysis, the calcium deposits were desorbed with 10% (w/v) cetylpyridinium chloride , and the absorbance at 562 nm was measured.After 14 days of adipogenic induction, lipid droplets were stained with Oil Red “O” . Cells were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, rinsed twice with PBS, and then stained with Oil Red “O” for 30 min. Images were captured under an inverted microscope .μg of total RNA with the PrimeScript RT Reagent Kit . Real-time PCR assays for collagen type I alpha 1 chain (Col1a1), alkaline phosphatase (Alp), runt-related transcription factor 2 (Runx2), osteopontin (Opn), osteocalcin (Ocn), adiponectin (Adipoq), CCAAT/enhancer-binding protein alpha (C/ebpα), fatty acid-binding protein 4 (Fabp4), peroxisome proliferator-activated receptor gamma (Pparγ), and perilipin 1 (Plin1) were performed on Roche Lightcycler 480 system with qPCR SYBR Green Master Mix  according to the manufacturers' instructions. The primer sequences for qRT-PCR are listed in β-Actin was used as the internal control gene. Data were analyzed by using the comparative CT method (2−∆∆CT) and expressed as fold changes respective to control.Total RNA was extracted by using RNA-Quick Purification Kit  as per the manufacturer's protocol. cDNA was synthesized from 1 μg) were then resolved on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. Nonspecific binding sites were blocked by incubating the membranes in bovine serum albumin (BSA) for 1 hour at room temperature. Then, the membranes were incubated overnight at 4°C with primary antibodies against the following: COL1A1 , ALP , RUNX2 , OPN , OCN , β-CATENIN , p-AKT , AKT , p-GSK-3β , GSK-3β , LAMIN B1 , and β-actin . After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody  for 1 hour at room temperature, and the immunoreaction signals were detected with the ECL reagent . For quantification, the intensity of each band was measured by the Image J software.Total proteins were extracted with RIPA lysis buffer  supplemented with 1% protease inhibitor cocktail  and 1% phosphatase inhibitors . Nuclear and cytoplasmic proteins were extracted using Nuclear and Cytoplasmic Protein Extraction Kit  according to the manufacturer's protocols. Protein concentration was quantified with a BCA protein assay kit following the manufacturer's instructions. Protein samples  overnight at 4°C followed by incubation with Alexa Fluor 594 Donkey Anti-Rabbit IgG secondary antibody . DAPI  was used to counterstain cell nuclei for 5 min at room temperature. Finally, fluorescence was visualized using confocal microscopy .Cells were seeded in confocal dishes at a density of 2 × 10p value < 0.05 was considered statistically significant. The SPSS 20.0 software  was used for all data analysis.All data were presented as the mean ± standard deviation (SD) of three independent experiments. Statistical difference was analyzed via one-way ANOVA. A As shown in −9, 10−7, and 10−5 M) for 2 days and then harvested for Annexin V/PI staining. Q1 (Annexin V–/PI+) represents necrotic cells, Q2 (Annexin V+/PI+) represents late apoptotic cells, Q3 (Annexin V+/PI–) represents early apoptotic cells, and Q4 (Annexin V–/PI–) represents live cells. The apoptosis rate was calculated as the percentage of early and late apoptotic cells. No significant difference in apoptosis rate was observed among these concentrations   for 1, 3, 5, and 7 days  treatment. ALP staining and ALP activity were significantly suppressed by DA after 7 days of differentiation  by qRT-PCR and levels of the corresponding proteins by western blot. Results showed that during osteogenic differentiation of rBMSCs, both mRNA and protein levels of these osteogenic marker genes were significantly reduced upon treatment with DA  treatment. Results showed that DA markedly suppressed lipid droplet formation and mRNA levels of adipogenic marker genes  in rBMSCs after 14-day induction , GSK-3β, phosphorylated GSK-3β (p-GSK-3β), and β-catenin in rBMSCs in response to treatment with DA . We found that the ratio of p-AKT/AKT, p-GSK-3β/GSK-3β, and β-CATENIN was reduced at 24 h after DA treatment. However, no significant change was observed in the total protein expression of AKT and GSK-3β  for 2 h. Interestingly, we found LiCl treatment rescued the inhibitory effects of DA (10−5 M) on ALP staining, ALP activity, and mineralized nodule formation in rBMSCs  and proteins  were also rescued by LiCl treatment  for 2 h. The results showed that LY294002 administration exacerbated the inhibitory effects of DA (10−5 M) on ALP staining, ALP activity, and mineralized nodule formation in rBMSCs   and D2-like receptors . The activation of D1-like receptors stimulates adenylate cyclase and subsequently increases the concentration of intracellular cyclic adenosine monophosphate (cAMP), while the activation of D2-like receptors exerts an opposite effect , 30. It −9, 10−7, and 10−5 M DA did not interfere with the apoptosis or proliferation of rBMSCs and significantly suppressed ALP staining and ALP activity in rBMSCs, which is known to be an early differentiation marker of osteogenesis. In the early stages of mineralization, ALP increases hydroxyapatite synthesis by producing inorganic phosphate, subsequently enhancing bone formation and mineralization [−9 and 10−7 M suppressed osteoblast mineralization of preosteoblast cell line MC3T3-E1 [−5 M) of DA has also been reported to inhibit osteogenic differentiation of BMSCs. However, in the same study, 5 × 10−9 M DA had an opposite effect on osteogenic differentiation [−5 M DA could promote osteoblast mineralization in MC3T3-E1 [β pathway, Wnt/β-catenin pathway, and ERK pathway [β/β-catenin signaling pathway.In the current study, we demonstrated that 10lization . In addiMC3T3-E1 . A highentiation . AnotherMC3T3-E1 . Such diMC3T3-E1 , 34. At MC3T3-E1 , 35. As  pathway –39. It w pathway , whereasβ, the best-characterized downstream target of AKT, is a pleiotropic serine/threonine protein kinase. Activated AKT further phosphorylates GSK-3β at Ser9 site, which leads to the inactivation of GSK-3β [β is involved in various intracellular signaling pathways, the most famous of which is Wnt/β-catenin signaling pathway. Studies have shown that GSK-3β is the connection between PI3K/AKT signaling pathway and Wnt/β-catenin signaling pathway, which is crucial to the stability of β-catenin in the cytoplasm [β-catenin in Wnt/β-catenin signaling pathway, GSK-3β can promote the degradation of β-catenin [β/β-catenin signaling pathway is involved in osteogenic differentiation and bone regeneration [β/β-catenin signaling pathway. Our results showed that DA downregulated the AKT phosphorylation, GSK-3β phosphorylation, and β-catenin expression levels. To further verify whether the AKT/GSK-3β/β-catenin signaling pathway was indeed involved in regulating the osteogenic potential of rBMSCs, AKT inhibitor LY294002 and GSK-3β inhibitor LiCl were utilized in the present study. Our results showed that treatment with LiCl rescued the inhibitory effects of DA on osteogenic differentiation of rBMSCs, while LY294002 exacerbated the inhibitory effects of DA on osteogenic differentiation of rBMSCs, which indicated that dopamine suppresses osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells via AKT/GSK-3β/β-catenin signaling pathway  showed that 5 × 10−5 M DA significantly promoted adipogenic differentiation of rBMSCs. However, the mechanisms of DA on adipogenic differentiation of rBMSCs require further investigation.AKT is a serine/threonine-specific protein kinase that plays a key role in many cellular processes, such as cell growth, survival, proliferation, apoptosis, differentiation, migration, transcription, and glucose metabolism , 41. Thef GSK-3β . GSK-3β ytoplasm , 46. As -catenin . Previouneration –51. Ther pathway . Besidesin vivo studies to support our conclusions, there is a need for further animal studies to investigate the effect of DA on bone remodeling. Further studies are required to fully elucidate the underlying mechanisms.Nonetheless, several limitations in our study should be noted to provide ideas for further research. First, we have not investigated which receptor is involved in the effect of DA on osteogenic differentiation. Second, as we did not conduct β/β-catenin signaling pathway is involved in this process. Therefore, our study may provide new insights for understanding the relationship between neurotransmitters and bone remodeling.In conclusion, our study demonstrates that DA suppresses osteogenic differentiation of rBMSCs and that AKT/GSK-3


Ki-67, as a proliferation biomarker for human tumor cells, was also assessed as an additional potential marker for differentiating KA from cSCC. In this report, these four indicators were analyzed in 42 KA and 30 cSCC cases with the use of the computer automated image analysis system. Computer automated image analysis is a time-based and cost-effective method of determining IHC staining in KA and cSCC samples. We found that C-Myc staining was predominantly localized in the nuclei of basal cells within KA patients, whereas cSCC staining was predominantly localized in the nuclei of diffuse cells. This C-Myc staining pattern has a sensitivity of 78.6% and a specificity of 66.7% for identifying KA. Moreover, positive rates of distinct expression patterns of C-Myc and Ki-67 may also serve as a means to clinically distinguish KA from cSCC. Taken together, our results suggest that these markers, in particular C-Myc, may be useful in differentiating KA from cSCC.The distinction between Keratoacanthoma (KA) and Cutaneous Squamous Cell Carcinoma (cSCC) is critical yet usually challenging to discriminate clinically and histopathologically. One approach to differentiate KA from cSCC is through assessing the immunohistochemical staining patterns of the three indicators,  Keratoacanthoma (KA) is a proliferative, fast-growing squamous epithelial skin tumor which shows a spontaneous regression. It most commonly occurs in the elderly, mainly in sun-exposed areas, with the face being particularly vulnerable, while multiforms and rash types are less common. According to the clinical manifestation of KA, the biological development of KA can be divided into proliferative, mature, and regressing stages. In all of these cases, there is a tendency to progress to a well-differentiated Cutaneous Squamous Cell Carcinoma (KA-cSCC) and KA with complete progression to cSCC .KA shares many clinical and histological features with the well-differentiated cutaneous condition of Cutaneous Squamous Cell Carcinoma (cSCC). In specific, the morphological and histopathological characteristics of KA are very similar to those of cSCC, making it difficult to distinguish between these two conditions. In fact, some clinicians consider KA to be a variant of cSCC , which cβ-catenin signaling pathway [β-catenin signaling pathway is closely associated with the development and metastasis of different tumors, including skin, colon, and breast cancers [β-catenin signaling pathway, β-catenin is essential for controlling hair follicle formation [β-catenin signaling pathway was functionally active during the growth phase of the KA tumor and that retinoic acid- (RA-) mediated Wnt inhibition was sufficient to induce spontaneous regression of KA [An important factor which can aid in such a differential diagnosis is the identification of specific, discriminatory clinicopathological biomarkers. Some clinicians have recommended that Ki-67 may possess this potential as it shows a positive percent of >20% in cSCC and ≤20% in KA . It has  pathway . Activat cancers , 13. In ormation –16 and tormation , 18. In on of KA .β-catenin signaling pathway in the development of cSCC and KA led us to conjecture that key signature molecules within this pathway may play different roles and show different expression patterns in KA versus cSCC. Accordingly, it may be possible to identify and isolate specific diagnostic biomarkers to assist in the differentiation of KA from cSCC. Given this potential, we selected critical molecules within the Wnt/β-catenin signaling pathway cascade, including β-catenin, C-Myc, and CyclinD1, for analyses  technology, which utilizes the immense computing power of computers to perform computer-aided diagnosis (CAD). CAD is a technique that can quantify the shape, size, and color information of pathology images, as well as enable image retrieval, pattern recognition, computer learning, and deep learning. In this way, it is possible to construct CAD mathematical models and extract corresponding quantitative features and/or identify a specific region of interest (ROI) within a pathology image. Such quantitative features or regions obtained with CAD represent potentially important tools to assist pathologists in quantitatively evaluating indicators to achieve a more rapid, accurate, and highly reproducible medical diagnosis –24. Our Surgical removal of skin KA (42 cases) and well-differentiated cSCC (30 cases) samples was performed at the First Hospital of China Medical University over the period from January 2016 to May 2021. None of these patients received radiotherapy, chemotherapy, and/or biological therapy prior to surgery. All cases were assessed and confirmed independently by two pathologists. Informed consent was obtained from patients according to the Declaration of Helsinki, and the Ethics Committee of the First Hospital of China Medical University approved these experiments.μm sections. The sections were deparaffinized and rehydrated using xylene and graded alcohol solutions. They were blocked with 2% bovine serum albumin and incubated with specific primary antibodies for 12 hours at 4°C, followed by incubation with a biotinylated secondary antibody. Subsequently, diaminobenzidine (DAB) was added dropwise over a 3- to 5-minute period and sections were then counterstained with hematoxylin. The primary antibodies included rabbit polyclonal antibodies of CyclinD1 , β-catenin , Ki-67 , and C-Myc .Formalin-fixed, paraffin-embedded sections were cut into 4 Images were generated using the Vectra™ 3.0 multiplexing image technology platform . The entire pathology section was scanned for 10x photos, then labeled using the Phenochart Whole Slide Viewer , with each tissue core scanned for production of 20x photos. The inForm® 2.4 image analysis software program  has a powerful self-learning feature enabling the identification and segmentation of specific tissue types through a “circle training” approach that allows for the measurement or scoring of biomarker expression in specific tissues, as well as within nuclear, cytoplasmic, and cytosolic regions. With this program, it is possible to convert subjective interpretational assessments of the pathology within sections into accurate and consistent objective morphological records.β-catenin and Ki-67 expressions in cSCC samples presented as examples and presented in A brief description of the computer software analysis steps is contained in t-tests, chi-squared tests, or one-way ANOVAs were used for these analyses. Graphs were generated using GraphPad Prism 9.0 software , Visio Drawing , and BioRender® software (USA) with BioRender® software publication agreement number VB23ZIU9YD. A P < 0.05 was required for results to be considered statistically significant.All data were analyzed using the SPSS 23.0 statistical software program . Results are presented as the means ± standard deviations (SD). Student's P = 0.000).A summary of the demographic and clinicopathological data of patients included in this study is contained in β-catenin in KA and cSCC patients was found to be located in cell membranes and/or the cytoplasm of the tumor island , while KA-cSCC, with Score 3+, was significantly greater than the KA-regressing subtype . Such results not only suggest a potentially significant effect of C-Myc but also suggest that Ki-67 and C-Myc may play similar roles in cSCC and KA. Based on previous diagnostic recommendations, we classified a Ki-67 positive percent of >20% as cSCC and that of ≤20% as KA [Interestingly, analyses of C-Myc and Ki-67 0% as KA , along wβ-catenin signaling pathway. CyclinD1 regulates the cell cycle primarily by boosting the G1/S phase transition, which then permits the initiation of cell division. Under normal conditions, CyclinD1 protein is only transiently present in the cell [In addition to C-Myc, CyclinD1 represents another major downstream target gene of the Wnt/the cell , howeverthe cell –35. Whilthe cell . Additioβ-Catenin was expressed in cell membranes and/or cytoplasm within both the KA and cSCC samples, with this expression in KA samples exhibiting a preference for cell membranes and some portions of the cell cytoplasm, while in cSCC samples, this expression was also found in the cell membrane but in relatively greater concentrations within the cytoplasm than that in KA, findings which were consistent with previous experimental results [β-catenin expression was slightly greater in cSCC versus KA, although these differences failed to achieve statistical significance. When KA subgroups with β-catenin Score 3+ were compared, levels of β-catenin expression were significantly greater in KA-regressing as compared with the other two subtypes. We should note that these findings may be somewhat questionable due to the limited sample size of KA-regressing specimens and a dearth of research available which could explain the morphological anomalies and underlying mechanisms. results , 37. Theβ-catenin, while activation of the Wnt/β-catenin signaling pathway increases the quantity of β-catenin in the “cytoplasmic pool” and its translocation to the nucleus, where it initiates and regulates transcription of downstream target genes [β-catenin signaling pathway in both KA and cSCC in this study, the significantly increased accumulation of free-form β-catenin within the cytoplasm of cSCC versus KA samples implies that more β-catenin would be available for entry into the nucleus. As compared with KA, cSCC demonstrates an increased ability to initiate and regulate transcription of downstream target genes and promote cell proliferation and differentiation. Moreover, cSCC has a greater capacity to control and activate the transcription of downstream target genes and enhance cell proliferation and differentiation.At rest, the cell has only trace levels of free et genes . As therβ-catenin, C-Myc, and CyclinD1 in tissue samples from KA and cSCC patients. These markers represent the major molecules within the Wnt/β-catenin signaling pathway that are believed to be involved in the development of KA and cSCC. In addition, Ki-67, a more established biomarker, was also evaluated in these samples. To evaluate these biomarkers, we used a computerized image platform, which provides a more objective, consistent, and reproducible method for analyzing these data than that achieved with a manual review. Based on results obtained with this method, we provide the first evidence indicating that C-Myc expression in KA was predominantly located in the nuclei of basal cells, whereas this expression in cSCC samples was predominantly in the nuclei of diffuse cells. In addition, we observed that distinct expression patterns of C-Myc and Ki-67, but not CyclinD1, may serve as a means to discriminate KA from cSCC during the clinical diagnosis process. Our findings also support the notion that KA's distinctive spontaneous regression is closely tied to the Wnt/β-catenin pathway in mediating hair follicle development. Based on these results, we propose that KA should be considered a separate mode of skin tumor development and carcinogenesis from that of cSCC.In this report, we investigated the expressions of