Polymeric hydrogels are a complex class of materials with one common feature—the ability to form three-dimensional networks capable of imbibing large amounts of water or biological fluids without being dissolved, acting as self-sustained containers for various purposes, including pharmaceutical and biomedical applications. Transdermal pharmaceutical microneedles are a pain-free drug delivery system that continues on the path to widespread adoption—regulatory guidelines are on the horizon, and investments in the field continue to grow annually. Recently, hydrogels have generated interest in the field of transdermal microneedles due to their tunable properties, allowing them to be exploited as delivery systems and extraction tools. As hydrogel microneedles are a new emerging technology, their fabrication faces various challenges that must be resolved for them to redeem themselves as a viable pharmaceutical option. This article discusses hydrogel microneedles from a material perspective, regardless of their mechanism of action. It cites the recent advances in their formulation, presents relevant fabrication and characterization methods, and discusses manufacturing and regulatory challenges facing these emerging technologies before their approval. Hydrogels are a collection of soft matter classes that include disparate varieties of polymeric materials from different origins that can form water-swollen three-dimensional networks via chemical or physical crosslinks in which the nature of the bonds formed determines the hydrogel reversibility. While there are a number of historical references to these materials—records documenting the use of the gel substances gelatin and aloe vera go back to Mesopotamia and ancient Egypt —the termSince the early development days, the popularity of hydrogels as carriers of physically, chemically, or biologically active ingredients has been continuously on the rise . Their vPharmaceutically, hydrogels can be utilized as standalone dosage forms or as parts of drug delivery systems (DDSs) and medical devices. They can be incorporated into drug-eluting DDSs either as the carrier matrix or by integrating them as stabilizers or release controllers ,10,11. SThe transdermal delivery route utilizes access through the skin, mainly using patches, gels, and emulsions, allowing patients to self-administer their medications without the systemic loss encountered via the oral route or the pain associated with injection—see Several physical and chemical solutions have been developed to overcome the stratum corneum barrier. Among the most promising options are the use of microneedle (MN) products .In 1967, Gerstel and Place filed the first percutaneous MN drug delivery device patent . After iMicroneedle arrays are micron-scaled (height ≤ 1000 µm with a tip diameter of 1–25 µm) projections assembled on a supporting base . MNs areMicroneedles are manufactured from a wide variety of materials, including glass, silicone, biodegradable polymers, and metals . In the From a material point of view, this classification system is restricting when describing hydrogel MNs since hydrogels could fit under both the dissolving and hydrogel-forming categories according to their components and properties.The application of hydrogels on skin paired with pre-treatment using MN arrays or commercial derma rollers was found to enhance their permeation of larger doses of actives. The application of thermogelling systems to pre-treated skin was found to fill the MNs’ created cavities, providing a sustained 72 h lasting release of methotrexate and fluorescein sodium ,40.The fabrication of hydrogel-based MNs combines physical microporation with the tunability of the residence time of hydrogels in one device.Hydrogels can be manufactured from a broad range of polymeric materials, which are commonly classified according to the origin of their material components  .This review aims to highlight recent advances in the emerging field of hydrogel-based MNs, summarizing the latest formulation trends, manufacturing methods, and performance evaluation techniques.The ideal drug delivery device should be able to (i) encapsulate active(s) in a biocompatible and mechanically robust system, (ii) allow the control of the release profile, and (iii) be easy to administer with minimal medical supervision . HydrogeHydrogel MNs are fabricated using a variety of methods and dried to obtain hard, solid MNs with sufficient mechanical properties for application. When embedded in the skin, the interstitial fluid hydrates the MNs via diffusion, and the dried matrix forms a hydrogel, which swells in place, imbibing large amounts of fluid withdrawn from its surroundings. The fluid movement within the polymeric network facilitates the API’s diffusion and release through the MNs’ created channels. For some systems, this could be followed by dissolution, depending on the matrix’s internal structure. The great swelling ability of MNs could be additionally utilized to absorb fluids to be used for diagnostic purposes.This article will discuss the most common hydrogel substrates used for MN systems from a material perspective, covering both dissolvable and swelling MN matrices. As highlighted in the recent publication by Moore et al. , hydrogeCellulose is a hydrophobic biodegradable polysaccharide linked by β- glycosidic bonds. It forms the main component of plant cells’ walls and can be produced by some bacteria. Its chemical structure can be modified  to produce a wide array of derivatives with diverse physicochemical properties that correlate with the degree of substitution (DoS). Popular pharmaceutical cellulose-based gelling agents include methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), ethyl cellulose (EC), hydroxyethyl cellulose (HEC), and carboxymethylcellulose (CMC), some of which display environmental sensitivities that could be exploited to control the MN release profile .Among the most widely used ones is CMC, a water-soluble anionic linear polysaccharide obtained by substituting some of the hydroxyl ends with carboxymethyl groups (DoS 1.2-2). The low cost and broad range of functionalities have seen the adoption of CMC for a diverse array of biomedical applications . CMCs haHigher concentrations of the low viscosity grades were found to give excellent results for the fabrication of systems prepared via droplet-borne air blowing (DAB) and centrifugal lithography (CL) processes that were employed in the manufacture of the promising lidocaine MN and typhus vaccine ScaA MNs ,52. SimiHPC and HPMC are nonionic water-soluble cellulose derivatives. Ito et al. compared the performance of three types of HPC lidocaine-loaded MNs in a topical cream. All the MNs showed a faster onset than the cream. The whole HPC-lidocaine-loaded MNs were more effective than only using loaded tips or the loaded MNs paired with a reservoir to deliver the drug, for which the presence of the reservoir suppressed the faster release observed for the other MN types . MNs conThe selection of a suitable cellulose-based agent depends on two factors. The first relates to the targeted gel (and MNs after drying) properties, which is affected by the degree of substitution and Mw. The second is the formulation recipe and whether the matrix charge could have detrimental effects leading to instabilities.Gram-positive and Gram-negative bacteria. This versatility allowed its use in various formats, ranging from injectables and topical productions to transdermal delivery [Chitosan (CS) is a linear cationic amino-polysaccharide composed of β--linked D-glucosamine and N-acetyl-D-glucosamine obtained via the alkaline deacetylation of chitin, the second-most-abundant natural biopolymer after cellulose. It is found in fungi, crustaceans, and insect shells. The biopolymer is soluble in dilute acidic solutions and can form stimuli-responsive gels at low concentrations when neutralized . CS is kdelivery . Its prodelivery . Studiesdelivery . CS gel delivery  and cetidelivery . Other adelivery , trifluodelivery ,65. Chemdelivery , as welldelivery .Hyaluronic acid (HA), also known as hyaluronan, is a liner anionic copolymer of non-sulfated glycosaminoglycan. It was extracted for the first time in 1934 from bovine vitreous fluid . HA is aThe hydrophilic nature of HA could pose challenges for fabricating systems to encapsulate hydrophobic loads or for those requiring slow release rates. Introducing stabilizers such as lipid-based nanoparticles (LNP), liposomes, and other polymers to HA matrices was recorded to overcome this limitation and enhance HA MNs’ performance ,77,78.Bombyx mori cocoons, which consists of two proteins: sericin and fibroin. Silk fibroin is generally regarded as safe (GRAS) by the FDA, and it is a biocompatible and biodegradable polymer [Silk is a fibrous natural polymer obtained from silkworm cocoons, spiders, mites, and pseudoscorpions’ silk fiber ,80,81. T polymer . Pure SF polymer . SF is b polymer . Pure SF polymer , which c polymer . Post-fa polymer .Zhu et al. compared three types of insulin-loaded SF systems: neat MN arrays, methanol-treated neat MNs, and MNs with a backing layer from proline amino acid. The authors reported that the neat MNs almost completely dissolved without undergoing any swelling, while the mixed-base systems experienced higher swelling and dissolution than the treated MNs—which barely released 40% of the payload after 9 h. In vitro tests showed that the mixed base system exhibited a faster release within the first two hours, and then the rate slowed down afterwards to achieve a complete release at around 9 h, providing a slower and smoother release profile when compared to insulin injections . SimilarPolyvinyl alcohol (PVA) is a water-soluble synthetic polymer prepared via the hydrolysis (saponification) of polyvinyl acetate. The length of the polyvinyl acetate units and the hydrolysis conditions can be controlled to produce a large variety of molecular PVA weights, in which the hydrolysis percentage is inversely proportional to the solubility . This isPVP is a synthetic nonionic water-soluble biocompatible polymer. The FDA-approved polymer is widely used as a plasma expander and in various medical applications due to its stability and good film-forming properties . PVP’s a® is either used as a stand-alone dissolving polymer matrix [® AN119—amphotericin B MNs [TM AN139 for formulations containing aminolevulinic acid and nadroparin calcium [TM S97 for the delivery of acyclovir and vitamin K [TM S97 were tested as carriers of the model protein antigen ovalbumin. The results showed that the uncross-linked systems demonstrated an enhanced immunological response and a peak release after 5 h, while the cross-linked MN matrix barely released 27% of the available load even after 24 h [Poly  is a synthetic-class biodegradable copolymer commercially available under the name Gantrez™. The relatively low-cost copolymer is widely used in cosmetics and personal care products . Four grr matrix  or crossr matrix . For thein B MNs , Gantrez calcium ,126, anditamin K ,128. Croitamin K ,130 or ditamin K ,133,134.ter 24 h .−1), double the value recorded for the medical-grade Tegaderm™ films. Therefore, this could negate the need for additional supporting adhesive strips for microneedle patch retention [As previously mentioned, HA has exceptional biocompatibility; however, it is not suitable for prolonged release purposes due to high water solubility . HA’s hyetention . The useetention . Chang eThe volume of the interstitial fluid (ISF) extracted was found to be inversely proportional to the UV exposure time without any correlation with the observed mechanical strength . Zheng eGelatin is a hydrophilic biopolymer obtained via the hydrolysis of collagen, a natural protein occurring in extracellular matrices . GelatinQiao et al. reported that their graphene-oxide-laden GelMA MN systems enabled the collection of large ISF volumes (21.34 μL in 30 min) and allowed the detection of multiple microRNA biomarkers to be used in psoriasis detection . The uniThe composition of the matrices in MN systems is usually investigated at various fabrication stages to ensure the uniformity of content and validate the effectiveness of crosslinking or polymerization reactions ,154,155.−1) covering the absorption radiation of most organic compounds. In their CS cetirizine-loaded MNs, Arshad et al. noticed that the positions of CS’s peaks  shifted to higher wavelengths in the dried MNs, suggesting a looser molecular packing and lower transition energies. The MNs’ spectra combined both CS and cetirizine peaks without any additional ones, indicating the absence of chemical interactions during casting [−1, attributed to the stretching of CH2 and formation of tertiary CH when compared to uncross-linked polymer [The most utilized method is Fourier-transform infrared spectroscopy (FTIR) due to its robustness, flexibility, and broad range  is heavily used to verify the degree of substitution in cross-linked GelMA and MeHA systems shown in Other spectral methods that have been reported in the literature include X-ray photoelectron spectroscopy (XPS), which is used to assess the MNs surface composition and detect any contamination attributed to molding, as reported for silicon residues on Gantrez AN-119 BF and amphotericin B MNs from polydimethylsiloxane (PDMS) casting .Thermal methods offer quick and easy gel quality control measurements. They allow the monitoring of stability and the concentration of the various systems’ components and can identify any thermally driven transitions that could affect fabrication or release. Popular methods include the following.Differential Scanning Calorimetry (DSC) measures the difference in the heat flow rate of a sample compared to a reference as a function of time during heating at a constant rate (with a linear temperature rise). The resulting thermal spectra can be used to characterize phase transitions, which provide information about the gel structure . For hydTGA is a thermal quantitative technique in which the substance mass is monitored continuously as a function of temperature or time in a controlled atmosphere to elucidate thermal stability. TGA uses a high-resolution mass balance to enable the detection of weight losses attributed to free and bound water , dehydraImaging techniques are regularly used to assess the geometric quality of MNs and indentation site inspection. The selection of the appropriate technique will depend on the purpose of imaging, whether it is quantitative or qualitative, the nature of the sample, and the required resolution. Common microscopic measurements are classified according to the illumination path and sensor type and include bright-field  and confMNs endure a spectrum of stresses, particularly during insertion, removal, general handling, and transportation ,166, so In many of the investigations, the mechanical strength of HG MNs has been assessed by compressing the entire patch using a flat, hard surface, such as a stainless steel plate or probe, as shown in With less frequency, researchers characterize the mechanical properties of individual microneedles. It is more challenging since it requires diminutive probes while ensuring the avoidance of contact with adjacent microneedles. In this regard, Du et al. (2021b) employed a glass rod measuring 100 µm in diameter, while Kim et al. (2013) employed a force sensor probe, and Lee et al. (2015) employed a stainless steel pillar; however, the detailed dimensional specifications of these probing instruments were not reported. An alternative approach involves sectioning samples to a size equivalent to that of a single microneedle, as demonstrated by Oh et al. (2022).2 where they touched the skin; they should all have demanded the same amount of force for insertion. This force was predicted to be around 0.058 N per needle.Researchers often use the minimum force of skin penetration as a parameter to validate the strength of MNs. A single value of 0.058 N/MN is used frequently in the literature ,137,175.A value of 0.1 N also often comes up in the literature. Yu et al. (2021) referred to it as the minimum force required to penetrate human skin. Wang et al. (2016) characterized it as the force required to puncture human skin. Zhu et al. (2019a) stated that the force needed to penetrate the skin is more than 0.1 N. An et al. (2022) referred to it as the force sufficient to insert into the skin. Qiao et al. (2022) used a similar value of 0.15 N/needle as a threshold value for the skin insertion of MNs. All these authors have made reference to the study conducted by Davis et al. in 2004, wherein a single MN was introduced into the skin of a human subject’s hand, specifically within a 1 cm² area at the base of a knuckle. The microneedles employed in this experiment had a height of 720 µm, a radius ranging between 30 and 80 µm, and a wall thickness spanning from 5 to 58 µm. The insertion process was carried out at a rate of 1.1 mm/s, utilizing a maximum force of 500 g. The recorded forces necessary for insertion ranged from 0.08 to 3.04 N. Consequently, it would be incorrect to assert that a minimum force of 0.1 N per needle is sufficient to breach human skin.Another study identified the force of insertion to be approximately 0.029 N/MN at an insertion speed of 0.5 mm/s and 0.021 N/MN at an insertion spend of 1 mm/s. MN arrays with varying numbers of MNs per array and different interspacing were inserted into an excised (700-µm-thick) and trimmed stillborn piglet’s skin .The typical method for evaluating the skin insertion ability of HG MNs involves applying pressure to MN samples onto the skin, followed by the removal of the sample. Staining agents such as trypan blue or methylene blue are then utilized to improve the visibility of the puncture sites. These staining solutions are applied to the skin for a period of 2 to 30 min, typically around 5 min. After this, any excess dye is wiped away, and the area where the MNs were applied is examined, as demonstrated in Of course, human or animal skin tissue is essential for assessing the skin puncture capability of HG MNs. While human skin is the most suitable for conducting experiments involving HG MNs or any other MNs, there are associated challenges. The utilization of human skin in scientific investigations necessitates endorsement from ethical and regulatory organizations, and there exist safety apprehensions concerning its manipulation. The procurement, preparation, and upkeep of human skin for testing can incur substantial costs and demand significant resources ,188. NevAlthough human skin is widely considered the standard for in vitro investigations into drug penetration, the challenges discussed in the previous paragraph mean that animal models can provide attractive alternatives. Porcine skin is recognized as the preferred animal model because it shares many anatomical, histological, and physiological similarities with human skin, and its penetration behavior is comparable .There are many reports of the ex vivo or in vitro introduction of MNs into porcine skin—as shown in Alternative animal skin models, such as those of mice, rats, or rabbits, have also been employed for HG MN penetration studies. When contrasted with pig skin models, utilizing rodent skin offers several benefits. The advantages of rodents are their compact size, straightforward handling, and relatively low cost . NotablyTM  folded eight times to create an approximately 1-mm-thick membrane demonstrated equivalent insertion profiles to those compared with neonatal pig skin. Since the thickness of each layer of ParafilmTM was approximately 100 µm, the total depth of penetration could be estimated by inspecting each layer for puncture marks [TM layer. Hence, it is recommended to assess the thickness of each layer before and after application.Several concepts of artificial skin models to study the penetration ability of MNs have been developed. A model introduced by Larrañeta et al. (2014) in which a sheet of Parafilmre marks . The advTM, agarose gel or PDMS can be used. Agarose typically originates from specific varieties of red seaweed and is a type of carbohydrate polymer. Agarose gel is considered a suitable skin model because it can be fine-tuned to resemble part of the stress–strain relation of human skin by varying the agarose concentration. MNs could be introduced into the agarose hydrogel directly or into a layered system consisting of an agarose hydrogel base with a Parafilm® layer placed on top, imitating the stratum corneum. This approach has been utilized to examine both drug release and needle penetration simultaneously [As an alternative to Parafilmaneously ,192,193.aneously ,195,196.Swellable microneedles, made from cross-linked hydrogels, expand in the skin without dissolving, facilitating ISF withdrawal and the controlled release of preloaded drugs ,197. The0 is the dry mass, and Wt is the mass of swollen MNs, respectively [Quantitively swelling is investigated by studying the mass change of MNs before and after incubation in PBS or insertion in tissue. The swelling ratio is calculated via the following equation:ectively ,137,138.The swelling rate, calculated using the same formula, involves periodically removing and measuring the mass of the microneedles ,152,166.Following the insertion of a dissolvable MN array into the skin, MNs dissolve upon contact with interstitial fluid, leading to the release of the drug cargo. Depending on the composition and dissolution method, HG MNs would be inserted into the skin or immersed in a PBS solution and then examined at designated time intervals to observe the presence or absence of the remaining needles on the microneedle patches using optical, digital, or stereo microscopy. The kinetics of dissolution can be investigated in vitro by submerging MNs in distilled water ,178, agaIn addition to ensuring that the dissolution process can be reliably replicated in vivo, it is essential to accurately determine the quantity of a drug administered. The amount of a drug actually delivered can often fall significantly below the maximum theoretical dosage because MNs may not completely dissolve. The majority of drug permeability studies utilize the Franz diffusion cell method. This method comprises two chambers: a donor chamber, where the tested formulation is applied with an animal model membrane positioned between it, and the receptor chamber, ensuring that the stratum corneum faces the donor compartment while the dermis contacts the receptor compartment. In vitro dorsal or abdominal rat skin ,138,149,After the microneedles have fully dissolved, it is necessary to select an appropriate method to determine the drug content based on the drug’s characteristics. Typically, for chemical substances, various methods are used, such as HPLC, fluorescence spectroscopy techniques, ultraviolet spectroscopy, and other methods relying on their physical and chemical properties for measurement ,173,187.Molding refers to any method that involves the replication of a master structure to produce MNs. The master mold is usually prepared from a metal or silicone substrate via any conventional tooling method capable of achieving the intricate MN geometry. Candidate techniques include micro-electro-discharge machining (µEDM) ,163,198,For solution mold casting, a reusable PDMS mold is created from a master mold. The PDMS mold is filled with the gel aliquots of the matrix (termed ‘drop casting’); the molds are usually vacuumed to remove air bubbles  and/or cAtomized spraying and piezoelectric (inkjet) dispensing offer alternative filling formats that lower the interfacial tension inside a mold and negate the need for the additional packing steps used in solution casting. They promise to enhance packing and enable precise dosing into cavities. McGrath et al. fabricated seven types of MNs, including ones using CMC, HPMC, and PVA, using an atomizer to dispense 10–50 μm droplets with 0.25-bar compressed air into a PDMS mold. Skin penetration and ketoprofen release were found to vary according to the formulation .Unlike spraying, piezoelectric dispensing has a higher targeting accuracy, and it allows the control of the volumes down to the picolitres level. Allen et al. demonstrated that the optimal influenza vaccine MNs’ biological activity depends on both the formulation and actuation settings used during dispensing .TM -based systems have been air-dried at room temperature, which is a considerably long process to obtain the optimal moisture levels [The MN cure conditions vary according to the materials used in a formulation. Chitosan- and Gantreze levels ,63,127. e levels , vacuum e levels ,192, mice levels , and visSurface drawing methods offer fast, mold-free fabrication that relies on the matrix’s adhesion, viscosity, surface tension and the movement of two flat surfaces to shape the MNs. Four types have been reported to be used for MN manufacturing: droplet-borne air-blowing (DAB), centrifugal lithography (CL), drawing lithography (DL), and electro-drawing (ED) .n = 20) clinical study reported on testing HA DAB-fabricated MNs. The system contained multiple actives with varied mechanisms of action , tranexamic acid, resveratrol, 4-n-butyl-resorcinol, and Halidrys siliquosa extract), and the multi-targeted approach was proven effective in treating melanogenesis [Droplet-borne air-blowing is performed at room temperature where two plates sandwich the hydrogel droplets, and the displacement of the two stacked plates draws the hydrogel droplets into biconcave-shaped pillars, which are solidified using blown air and then separated to produce MNs at each plate . Kim et ogenesis .For centrifugal lithography, the plates are loaded vertically into a centrifuge that elongates the drops and induces the hydrogel drops into shape via centrifugal evaporation. Once the structure is thinned enough, the MNs are solidified via the application of low temperatures that could be paired with vacuum . Two grog), and curing is achieved by reducing the temperature below its Tg [Drawing lithography uses a similar drawing technique to the methods previously discussed, with one difference: the matrix material is only drawn at its glass transition temperature (Tw its Tg . DL fabrw its Tg .Electro-drawing is a contact-free fabrication method performed at mild temperatures (20–40 °C) in which the gel droplet is drawn to be shaped from the plate through the application of an electro-hydrodynamic force; then, heat is applied to evaporate the solvent and solidify the MNs. ED is not widely used and has only been used to fabricate MNs from Poly(lactic-co-glycolic acid) ,220. Alt® AN 169 MNs with miconazole [w/v) MNs were simultaneously UV-cured during the printing process and air-dried at room temperature to obtain the final product. The authors reported that the recorded tip sharpness and displacement forces were suitable for penetration. Moreover, the printed MNs showed promising in vitro release and antimicrobial activity levels [Additive manufacturing (AM) has been touted as a promising manufacturing method for a myriad of MN types due to its high resolution, flexibility, and reasonable production costs ,222. Howconazole . DLP wasy levels . Visibley levels .The development of gel-based MN technologies still suffers from a multitude of challenges that need to be addressed for society to benefit from these technologies’ full potential. These challenges can be addressed using a three-pronged approach to ensure optimal performance and wide adoptability.Regardless of the final MNs’ applications, the materials used need to be biocompatible and suitable for biomedical applications. Further studies will need to be performed on a formulation-by-formulation basis to elucidate the effects of the local prolonged use of the polymers used with a particular focus on their metabolism and excretion routes . The MN TM dissolving MNs that contained 30% w/w of ibuprofen, a low-potency active. The highly filled MNs still achieved 90–100% insertion rates, and the ibuprofen plasma levels in rats after 24 h were 20 times greater than the human therapeutic level [A 160-day trial was performed to evaluate the effects of administering daily PVA MNs . The dorsal-skin bright-field images showed complete recovery within 60 min, and the hematologic and histological results indicated the absence of any toxicity in healthy female mice . Gel-basic level . HoweverFabrication challenges significantly depend on the gel properties and intended use of MN systems; however, common issues for hydrogels are scale-up issues such as ensuring material consistency, process repeatability, and the resulting microneedle quality for high-volume manufacturing runs whilst simultaneously ensuring dose uniformity and sterility, which will be critical for regulatory approval. Any of the methods discussed in According to the ICH Q1A(R2) stability guidelines for drug products, thermal stability and moisture sensitivity should be tested for periods long enough to cover storage, shipment, and application . For dehHiraishi et al. reported that HA MNs’ mechanical failure force was inversely proportional to the RH and that its values should be maintained within 11–75% . HoweverFor the industrial-scale production of MNs, a good manufacturing practice (GMP) framework and standardized testing procedures need to be developed to ensure quality and performance, but cost optimization should also be a focus to drive down production costs to a point at which MN devices will be readily accepted in the marketplace. These procedures remain difficult to develop in the absence of regulatory requirements. All medical MN systems aim to penetrate the stratum corneum, therefore exceeding the legal restriction for the FDA’s Class I medical devices. According to the FDA’s latest regulatory considerations published in 2017 and revised in 2020, MN devices fall under Class II medical devices, with the possibility of granting De Novo requests and taking into consideration the intended use. The World Trade Organization and European Regulation followed the FDA’s lead in classifying the devices as Class II  ,240. As TM and chitosan, have intrinsic antimicrobial qualities, with the latter being angiogenic as well; this, however, does not guarantee sterility during application [TM S-97, PEO (Mw of 10 kDa), and PVA (Mw of 58 kDa) on mice led to no changes in the skin’s barrier functions. Additionally, the authors reported that infection and inflammation biomarkers remained statistically indifferent from the control over the span of the five-week study, regardless of the formulation, MN density, or number of applications [Skin irritation is a natural, unavoidable immunogenic side effect of the application of MN systems. It appears visually as redness paired with minor swelling in the area, indicating the formation of local erythema. These temporary effects could facilitate the activity of some drugs, and MNs sensitivity is routinely assessed via pharmacodynamic/pharmacokinetic activity during clinical studies. Irritation can be minimized by adjusting the formulation and optimizing the design parameters . Patientlication ,250. A slication . The repications . More reications .Only a few transdermal MNs have been registered in phase 2 and 3 clinical trials; none, however, were completed. Radius Health announced in 2021 that its abaloparatide MN system (wearABLe) trial did not meet its primary or secondary endpoints and ceased all work and development on the system in June 2022 . Around The standardization of the testing and clinical assessment process could significantly enhance the chances of MN systems’ approval. As previously discussed and evident to any reader soon after delving into the MN literature, there is no universal evaluation process of MN performance, and only a few authors have pointed to their methods’ limitations in the process, which contributes to many of the challenges encountered. A fundamental understanding of the materials’ behaviors and how to quantify them is essential to develop these standard guidelines in order to optimize both the fabrication methods and end products.For example, dehydrated MNs’ mechanical assessments depend on many factors beyond the arbitrary force value. These factors include the experimental setup, the nature of the polymeric material, the tissue type, its state—its hydration or tension levels—and the MN application method. Although there is a direct correlation between the dried material property and its starting gel/solution rheological behavior, these measurements are rarely seen in the hydrogel-based MN literature. If conducting similar studies becomes customary, it could help avoid unsuitable fabrication techniques or formulations to enhance the quality of produced MNs.The emerging field of MN delivery systems is steadily growing, and new technologies are continuously being developed to enhance formulation stability, mechanical performance, and biological effect.The versatility and unique properties of hydrogels make them ideal systems for biomedical applications. When used in transdermal microneedle systems, they can provide mechanical strength for insertion, a carrier for preserving the API, and a controlled release to match the intended dose profile.Regulatory uncertainties have impeded the routes to market. However, advances in formulation and manufacturing technologies are providing improved devices that are now demonstrating regulatory compliance. New guidelines for designers and manufacturers of hydrogel MN systems will be soon implemented, which will encourage further industrial adoption and provide new routes for the transdermal delivery of a broad range of actives.


The Wnt/β-catenin signaling pathway is a classical Wnt pathway that regulates the stability and nuclear localization of β-catenin and plays an important role in adult heart development and cardiac tissue homeostasis. In recent years, an increasing number of researchers have implicated the dysregulation of this signaling pathway in a variety of cardiac diseases, such as myocardial infarction, arrhythmias, arrhythmogenic cardiomyopathy, diabetic cardiomyopathies, and myocardial hypertrophy. The morbidity and mortality of cardiac diseases are increasing, which brings great challenges to clinical treatment and seriously affects patient health. Thus, understanding the biological roles of the Wnt/β-catenin pathway in these diseases may be essential for cardiac disease treatment and diagnosis to improve patient quality of life. In this review, we summarize current research on the roles of β-catenin in human cardiac diseases and potential inhibitors of Wnt/β-catenin, which may provide new strategies for cardiac disease therapies. Canonical Wnt/β-catenin is primarily involved in the regulation of cell fate decisions and proliferation during embryonic development and tissue homeostasis in adults, while non-canonical pathways mainly regulate the polarity, mobility and migration of the cell , frizzled (Fzd), dishevelled (Dvl), GSK-3β, Casein kinase 1α (CK1α), Axin, Adenomatosis Escherichia coli (APC), Protein phosphatase 2A (PP2A), E3-ubiquitin ligase β-TrCP, β-catenin, and T-cell factor/lymphoid enhancer factor (TCF/LEF). Wnts are secreted lipoglycoproteins. Nineteen Wnt proteins have been identified in mammals. Among them, Wnt1, Wnt3a, Wnt7a, Wnt7b, Wnt 8 and Wnt 10a act as ligands to activate the canonical Wnt/β-catenin pathway . Wnts re factors . In addi factors . Interes factors .via β-TrCP. As a result, β-catenin is maintained at a low level in the cytoplasm and nucleus. In the presence of Wnt signals, the Wnt protein binds to the extracellular N-terminal domain under the synergistic action of LRP5/6. During this process, FZD is homodimerized, and FZD-LRP5/6 is heterodimerized. Subsequently, Dvl proteins in the cytoplasm are recruited to the cell membrane and further oligomerized. DvL contains three main domains: the N-terminal DIX domain, central PDZ domain and C-terminal DEP domain. Dvl oligomerization recruits AXIN. Therefore, AXIN-bound GSK3β and CK1α can get close to LRP5/6 and phosphorylate it. Phosphorylated LRP5/6 can provide an AXIN binding site  is a cardiac disease with high morbidity and mortality. Acute MI is an event associated with myocardial necrosis caused by ischemia and hypoxia in the coronary arteries. MI is usually accompanied by oxidative stress, inflammatory responses and the transformation of cardiac fibroblasts to cardiac myofibroblasts . The Wntin vitro model of oxidation-damaged cardiomyocytes. Reactive oxygen species (ROS) can activate phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT) by inhibiting phosphatase and tensin homolog deleted from chromosome (PTEN). Phosphorylated AKT was shown to activate the Wnt/β-catenin pathway by phosphorylating GSK-3β , which effectively prevents cardiac rupture. β-Catenin plays an important role in myocardial fibrosis. It was found that overexpression of β-catenin could upregulate various fibrosis markers in fibroblasts . MoreoveIt was also reported that β-catenin overexpression could not only induce fibroblast DNA replication and increase the number of fibroblasts but also induce the differentiation of fibroblasts into myofibroblasts . Severalin vivo , the α subunit of the myocardial voltage-gated sodium channel. Rong Huo et al. (Tbx3 (a suppressor of Scn5a expression) promoter at -871 bp, directly activates Tbx3 transcription, and directly inhibits Nav1.5 expression (Scn5a promoter to inhibit the expression of Nav1.5 (+/K+ channel genes were not different between WT (wild type) and KO mice  β-caf Nav1.5 . The inhf Nav1.5  observed KO mice .PKP2), desmoplakin (DSP), desmoglein-2 (DSG2), democollin-2 (DSC2) and plakoglobin (JUP) (Arrhythmic cardiomyopathy (ACM) is an inherited disease characterized by fibrofatty replacement of the ventricular myocardium. Although the incidence is low (from 1/2000 to 1/5000 people worldwide), it can lead to life-threatening arrhythmias and sudden death, especially in young people and athletes, and there is currently no effective treatment for ACM . Approxiin (JUP) . DesmosoDSP) can cause telomere damage, leading to plakoglobin (PG) dissociation and nuclear localization. PG and β-catenin have a high degree of sequence identity and homology, and they are usually antagonistic on multiple levels  was associated with the Wnt/β-catenin signaling pathway. β-catenin and AnkB are molecular chaperones, and AnkB is required for the normal localization of β-catenin  is a major cardiovascular complication associated with diabetes that is independent of hypertension, atherosclerosis and other cardiac diseases. DCM is characterized by hypertrophy and myocardial dilatation, as well as abnormal cardiac structure and function, and eventually develops into heart failure. The pathogenesis of DCM remains incompletely understood. It is believed to be related to oxidative stress, inflammation, myocardial apoptosis, autophagy and mitochondrial damage . Many reMyocardial hypertrophy (MH) is an adaptive change in the myocardium induced by various external stimuli. The markers of MH include myocardial cell volume enlargement and the expression of fetal genes, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (αMHC) . Studiesin vivo to treat some cardiac diseases. The effects of several Wnt/β-catenin pathway inhibitors on cardiac diseases are shown in To date, many researchers have made efforts to develop ideal Wnt/β-catenin pathway inhibitors for the treatment of certain diseases. As a result, many potential therapeutic inhibitors have been developed, including natural and synthetic proteins, antibodies and small molecule compounds. These inhibitors mainly act by inhibiting Wnt or β-catenin activity. At present, some inhibitors have been studied The Wnt/β-catenin signaling pathway is a complex protein network that is involved in various physiological and pathophysiological processes from cardiac development to cardiac muscle remodeling and myocardial repair. Without Wnt, β-catenin does not accumulate in the cytoplasm because there is a destruction complex that typically degrades it. However, under certain circumstances, the Wnt/β-catenin pathway is overactivated, which promotes more β-catenin entry into the nucleus and initiates the expression of downstream target genes . Recentlin vivo.Therefore, exploring inhibitors against the Wnt/β-catenin signaling pathway is considered a novel method for the treatment of cardiac diseases. The discovered inhibitors act mainly by binding directly to Wnt, inhibiting Wnt palmitoylation and secretion and blocking the β-catenin/CBP protein interaction. These inhibitors have been shown to be effective in treating some cardiac diseases In summary, this review provides a detailed summary and analysis of recent studies on the role of β-catenin in cardiac diseases and some potential inhibitors of Wnt/β-catenin. The expression of β-catenin may be a useful biomarker for identifying patients who may best respond to anti-Wnt/β-catenin therapy in cardiac diseases. Even so, the reason that cardiac diseases affect activation of the Wnt/β-catenin signaling pathway and the molecular mechanisms of Wnt/β-catenin involved in cardiac disease progression need to be further studied. Further study and investigation of the regulatory mechanism of the Wnt/β-catenin signaling pathway and the role of inhibitors will provide new ideas for the early diagnosis and precise treatment of related cardiac diseases.


Tppp3-positive cells in the dermis of the paw is associated with the development of fibrosis. Fibrosis development and progression via Wnt/β-catenin signalling are closely related to stromal cell–macrophage interactions, and Wnt/β-catenin signalling activation in Tppp3-positive stromal cells causes M2 macrophage infiltration via chemokine Cxcl14, resulting in the formation of a TGF-β-expressing fibrotic niche. Inhibition of Cxcl14 mitigates fibrosis by decreasing macrophage infiltration. These findings suggest that Cxcl14-mediated stromal cell–macrophage interaction is a promising therapeutic target for Wnt/β-catenin-induced fibrosis.Dupuytren’s contracture, a superficial dermal fibrosis, causes flexion contracture of the affected finger, impairing hand function. Specific single-nucleotide polymorphisms within genes in the Wnt signalling pathway are associated with the disease. However, the precise role of Wnt signalling dysregulation in the onset and progression of Dupuytren’s contracture remains unclear. Here, using a fibrosis mouse model and clinical samples of human Dupuytren’s contractures, we demonstrate that the activation of Wnt/β-catenin signalling in  Wnt/β-catenin signalling activation in the dermis cause fibrosis by cell-autonomous program and by a non-cell-autonomous program mediated by TGF-β niche formation based on Cxcl14-mediated interactions between stromal cells and M2 macrophages. Dupuytren’s contracture represents a prevalent condition, particularly among Western populations, with an estimated prevalence of 21% by the age of 65 years2. Although a collagenase Clostridium histolyticum injection is available as a minimally invasive therapy, surgical excision remains a reliable treatment method for this disease3. However, surgical complications, including hematomas, nerve injuries, and wound healing complications, are frequently observed, with incidences of 23%4.Dupuytren’s contracture is a superficial dermal fibrotic disease in the hand that causes flexion contracture of the affected fingers and impairs hand function. Genetic predisposition, ethnic characteristics, and other factors, including alcohol intake, smoking, diabetes, repetitive trauma, and exposure to vibration, have been identified as risk factors for Dupuytren’s contracture development, suggesting that it is a multifactorial disease5. Recent genome-wide studies have identified single-nucleotide polymorphisms (SNPs) in a subset of genes involved in the Wnt signalling pathway, including WNT2, WNT4, WNT7B, SFRP4, RSPO2, and SULF17. β-catenin, a downstream target of canonical Wnt signalling, is expressed in most myofibroblasts in the nodule, accumulating in their nuclei8. Therefore, the involvement of such Wnt signalling-related SNPs in Dupuytren’s contracture has been investigated11. The expression of WNT7B, a canonical Wnt ligand, was increased in Dupuytren’s nodules compared with that in unaffected palmar aponeurosis10. Moreover, its risk genotype was associated with Dupuytren’s contracture development11. The mRNA expression of SFRP4, a Wnt antagonist, was increased in nodules compared with that in unaffected palmar aponeurosis in Dupuytren’s patients9. However, its protein secretion decreased in Dupuytren’s tissues with a high-risk genotype compared with that in those with a low-risk genotype7. Thus, the Wnt signalling pathway is likely implicated in Dupuytren’s contracture.Myofibroblasts, the predominant stromal cells in Dupuytren’s contracture, play a central role in fibrosis by producing excessive extracellular matrix and pathological collagen14. Mast cells and macrophages, especially alternatively activated macrophages (M2), are involved in the fibrotic mechanism via chemotactic factors14. However, the role of Wnt signalling activation in the development and progression of Dupuytren’s contracture remains to be elucidated. Moreover, additional research is required to investigate the role of Wnt signalling in the interaction between stromal and immune cells.Recent studies revealed the possible involvement of stromal cell–immune cell interaction in myofibroblast differentiation and its activation in Dupuytren’s contractureTppp3)-positive cells resulted in the development of fibrosis of the paw, and its progression was markedly dependent on Wnt/β-catenin signalling activity. However, in addition to Wnt/β-catenin signalling-dependent myofibroblast differentiation in a cell-autonomous manner, fibrosis development and progression via Wnt/β-catenin signalling were closely related to stromal cell-immune cell interactions marked by M2 macrophage infiltration. Furthermore, transforming growth factor-β (TGF-β) was highly expressed in the fibrotic niche, consisting of stromal cells and macrophages, causing myofibroblast differentiation of stromal cells. Notably, the activation of Wnt/β-catenin signalling in Tppp3-positive cells upregulated the expression of chemokine C-X-C domain ligand 14 (Cxcl14), which is involved in macrophage infiltration. Furthermore, the blockade of Cxcl14 mitigated Wnt/β-catenin-induced fibrosis of the paw by decreasing macrophage infiltration.The aim of this study was to investigate the relationship between Wnt signalling activation and the development of dermal fibrosis using a fibrosis mouse model and human clinical samples of Dupuytren’s contractures. Our analyses revealed that activation of Wnt/β-catenin signalling in dermal tubulin polymerization-promoting protein family member 3  mice and confirmed that many tdTomato-positive cells were localized in the dermis of the paw  -inducible β-catenin-expressing mice (in) Fig. . Similarnth Fig. . Dox admnth Fig. . Conversnth Fig. . These fAxin2, a target gene of β-catenin 18, type I collagen , and type III collagen (Col3a1)  Fig. . Conversa1) Fig. , which i20. Neither inhibitor decreased the expression levels of myofibroblast markers , nor did they impact the expression of collagens (COL1A1 and COL3A1) , and MSAB, which degrades β-catenin directlyTppp3-lineage cells (β-catenin-induced cells) and non-Tppp3-lineage cells (β-catenin-non-induced cells), indicating that a non-cell-autonomous mechanism may also participate in fibrosis development induced by Wnt/β-catenin signalling activation  were present in fibrotic regions whose formation was induced by Wnt/β-catenin signalling activation  macrophages, with Tgfb1 as the predominant isoform  and compared their phenotypes with those of Tppp3-CreERT2/β-catenin ex3flox mice (Tgfbr2 wt mice) at 1 month after tamoxifen administration  a cell-autonomous fibrotic program induced by Wnt/β-catenin signalling activation in Tppp3-positive cells; and (2) a non-cell-autonomous fibrotic program mediated by TGF-β, which was released based on the interaction between Tppp3-positive stromal cells and macrophages via the chemokine Cxcl14, induced by Wnt/β-catenin signalling activation in Tppp3-positive cells  ameliorated bleomycin-induced murine dermal and lung fibrosis and chronic hepatitis C virus-induced liver fibrosis in vivo32. In contrast, some studies have reported that the expression of αSMA, a typical myofibroblast marker, was not directly induced by activation of Wnt/β-catenin signalling via Wnt3A34. Our study showed that the myofibroblast phenotype was directly induced by Wnt/β-catenin signalling in mice but not in humans. Verjee et al.13 demonstrated that tumour necrosis factor (TNF) treatment for palmar dermal fibroblasts derived from patients with Dupuytren’s disease led to activation of Wnt/β-catenin signalling through GSK-3β phosphorylation and inhibition, resulting in upregulated COL1 and α-SMA expression. Meanwhile, activation of Wnt/β-catenin signalling by a GSK-3β inhibitor (SB-216763) did not induce COL1 and α-SMA expression. These findings suggest that the role of Wnt/β-catenin signalling in fibrosis might differ across cell types, biological species, and fibrosis models. Moreover, in addition to Wnt/β-catenin-mediated cell-autonomous myofibroblast differentiation, alternative mechanisms may exist for fibrosis development and progression.Wnt/β-catenin signalling is an important pathway in organ development and cellular differentiation35. All three TGF-β isoforms induce a fibrotic response, although the role of TGF-β3 as a fibrosis inducer or repressor remains controversial36. Interestingly, TGF-β stimulated Wnt signalling by inducing the nuclear accumulation of β-catenin in rat pulmonary alveolar cells and human fibroblasts38 and induced αSMA expression by interacting with Smad3, CBP, and β-catenin38. Therefore, β-catenin is essential for TGF-β-mediated fibrosis. Similarly, we showed that TGF-β1 treatment induced nuclear translocation of β-catenin in human Dupuytren’s contracture-derived cells. Myofibroblast differentiation via Wnt3A was observed in the presence of TGF-β34. Our in vivo analysis using Tppp3-CreERT2/β-catenin ex3flox/Tgfbr2flox/flox (Tgfbr2-KO) mice demonstrated that TGF-β signalling was associated with Wnt/β-catenin-induced fibrosis. Together, these findings suggest that Wnt/β-catenin signalling and TGF-β signalling are intimately related to fibrosis and could be potential therapeutic targets for fibrotic disease. However, in this study, Wnt signal inhibitors PNU-74654, MSAB, and XAV939 did not reduce the expression of myofibroblast markers (including αSMA) and collagen in human Dupuytren’s contractures. Moreover, using TGF-β inhibitors for molecular therapy is challenging owing to the crucial role of TGF-β in the maintenance of cell homeostasis22. Thus, targeting these signals directly in the treatment of Dupuytren’s contractures may prove difficult.TGF-β is closely associated with fibrosis and is a representative fibrosis inducer39. Chronic inflammation by immune cells (particularly macrophages) is involved in fibrosis development in several organs and tissues, including pulmonary fibrosis, skin fibrosis, and liver fibrosis41. Dupuytren’s contractures share this pathological process of the development and progression of fibrosis14. Izadi et al.12 reported reciprocal activating pathways through stromal cells-expressed IL-33 and TNF-expressing M2 macrophages and mast cells. Furthermore, they demonstrated that anti-IL-33 and TNF receptor II antibody treatment downregulated the expression of fibrotic genes and reduced cellular contractility of Dupuytren’s myofibroblasts. Akbar et al.14 showed that mast cell-expressed IL-13 activated myofibroblasts through STAT signalling and that tofacitinib, an inhibitor of JAK1 and JAK3 related to the phosphorylation of STAT1 and STAT6, reduced proliferation and fibrotic gene expression in Dupuytren’s myofibroblasts. Thus, it is expected that the stromal cell–immune cell interaction can serve as a novel therapeutic target for Dupuytren’s disease.Recently, the importance of stromal cell–immune cell interactions has been recognized in fibrotic disease development and progressionTppp3-positive cells recruited macrophages and formed a TGF-β-expressing niche via Cxcl14. Macrophages are classified into two subtypes: classically activated macrophages (M1) and alternatively activated macrophages (M2). TGF-β is predominantly expressed in M2 macrophages, and its expression in these macrophages increases when they are in contact with fibroblasts42. Macrophage differentiation and recruitment are induced by several kinds of cytokines and chemokines47. Cxcl14/CXCL14 is the most recently identified C-X-C chemokine and is highly conserved in mice and humans48. Cereijo et al. 43 and Wang et al. 47 demonstrated that Cxcl14, secreted by brown adipocytes and pericytes, recruits macrophages and differentiates them into the M2 subtype, indicating that it could be an inducer of M2 macrophage polarization. Here, we demonstrated that Cxcl14/CXCL14 expression was upregulated via Wnt/β-catenin activation in murine dermal Tppp3-positive cells and human Dupuytren’s contracture-derived cells. In addition, TGF-β1 upregulated Cxcl14/CXCL14 expression in murine dermal fibrotic cells and human Dupuytren’s contracture-derived cells. Thus, a TGF-β-expressing niche, induced by Wnt/β-catenin signalling activation, may be stabilized via a positive-feedback interaction between stromal cells and macrophages through Cxcl14/CXCL14.In this study, we found that Wnt/β-catenin signalling activation in Cxcl14 is involved in the progression of murine liver fibrosis49. However, no study thus far has analysed its function in disease development and progression. Notably, knockdown of Cxcl14 expression and neutralization of Cxcl14 via antibodies ablated macrophage migration caused by the senescence-associated secretory phenotype both in vitro and in vivo, respectively50. Consistent with this report, we revealed that the neutralization of Cxcl14 in Tppp3-CreERT2/β-catenin ex3flox mice reduced M2 macrophage infiltration and mitigated dermal fibrosis in vivo. Moreover, CXCL14 is highly expressed in human idiopathic lung fibrosis51, and its expression was observed in Dupuytren’s contractures52, suggesting that it may also be associated with macrophage infiltration in human diseases.53. This limitation may have been responsible for the differences in myofibroblast differentiation and induction of TGF-β expression in response to Wnt/β-catenin signalling activation between mice and humans in this study. Therefore, to analyze the development and progression of Dupuytren’s contracture smore accurately, it may be necessary to create alternative mouse models, focusing on known SNPs in Dupuytren’s contractures7.This study has some limitations. First, we generated a mouse fibrosis model with β-catenin gene mutations; however, β-catenin gene mutations are absent in human Dupuytren’s contracturesSecond, Cxcl14 inhibition did not completely prevent M2 macrophage infiltration and fibrosis development; therefore, other factors controlling stromal cell–immune cell interaction could be involved in Wnt/β-catenin-induced fibrosis. Furthermore, Wnt/β-catenin signalling-induced fibrosis involves both cell- and non-cell-autonomous fibrosis programs Figs. , and 8. Cxcl14/CXCL14 expression in Tppp3-positive cells and human Dupuytren’s contracture-derived cells. Specifically, luciferase reporter constructs containing sequences spanning from +1908 to −292 relative to the start codon of murine Cxcl14 failed to upregulate promoter activity upon Dox-inducible β-catenin expression and TGF-β1 administration (see Supporting Data). These results suggest the possible existence of other regulatory mechanisms influenced by β-catenin and the TCF/LEF complex or suggest indirect regulation mediated by β-catenin-target genes. Moreover, we did not reveal the precise mechanisms by which Cxcl14 regulates TGF-β expression in M2 macrophages. Additionally, most of its receptors, with the exception of CXCR4 and IGF-1R55, remain elusive48, leaving the exact downstream pathway unclear. Further analyses are required to investigate the clinical application of Cxcl14-associated pathway-targeted therapy.Third, we did not elucidate the precise mechanism through which Wnt/β-catenin signalling regulates 57. In addition, Cxcl14-deficient mice exhibited lower weight, high blood glucose levels due to insulin resistance, and reduced bacterial clearance compared with wild-type mice, although the mice were viable58. Therefore, we should validate the risk of side effects due to blocking Cxcl14 and overcome them for clinical application.Fourth, Cxcl14 may play a role in the regulation of the central nervous system, specifically in feeding behavior and host defense against infectionsCxcl14-mediated cellular interaction between stromal cells and macrophages via activation of Wnt/β-catenin signalling and the formation of a TGF-β niche reflects an important process in the development and progression of fibrotic diseases. This mechanism of cellular crosstalk between stromal cells and macrophages could be a promising therapeutic target for fibrosis, including Dupuytren’s contracture.Collectively, identifying Experiments using clinical samples of human Dupuytren’s contracture were approved by the Ethics Committee of Gifu University . Between 2016 and 2023, the patients underwent surgery for Dupuytren’s contracture in our institution and were informed of this research. We obtained written informed consent from 14 patients (13 men and 1 woman) who agreed to this research, and excised samples were collected at the time of the operation. The patients were as follows: DD-1, 72-year-old female; DD-2, 86-year-old male; DD-3, 76-year-old male; DD-4, 76-year-old male; DD-5, 71-year-old male; DD-6, 75-year-old male; DD-7, 51-year-old male; DD-8, 80-year-old male; DD-9, 71-year-old male; DD-10, 86-year-old male; DD-11, 75-year-old male; DD-12, 70-year-old male; DD-13, 79-year-old male; and DD-14, 55-year-old male. All ethical regulations relevant to human research participants were followed.Rosa26-LSL-tdTomato (Ai9), Rosa26-M2rtTA, and Rosa26-LSL-rtTA-ires-EGFP mice were purchased from Jackson Laboratory (https://www.jax.org/). Tppp3-CreERT2 mice and doxycycline-inducible constitutive active β-catenin (S33 mutation) mice (Col1a1::TetO-β-catenin) were previously established60. β-catenin exon3 floxed mice (β-catenin ex3flox)62 and Tgfbr2 floxed mice (Tgfbr2flox)64 were kindly gifted by Makoto M. Taketo and Harold L. Moses, respectively. For the tamoxifen-inducible Cre-recombination experiment, 1-month-old mice were treated with 1 mg of tamoxifen (Sigma–Aldrich) or corn oil as a control (Wako) for three consecutive days. For doxycycline -inducible β-catenin induction, the mice were treated with Dox-containing water at 50 µg/mL for 1–2 months after Cre-recombination. Both male and female mice were randomly used without bias.All animal experiments were approved by the Gifu University Animal Experiment Committee , and the care of the animals was implemented following the Animal Research: Reporting of in Vivo Experiments guidelines. Tppp3-CreERT2/β-catenin ex3flox) and Dox-inducible β-catenin-expressing mice (Tppp3-CreERT2/Rosa26-stop-tdTomato/Rosa26-stop-rtTA/Col1a1::TetO-β-catenin) under a surgical microscope and dissociated with collagenase for 30 min at 37 °C. Next, they were cultured in a 12-well plate (Thermo Fisher Scientific) with Dulbecco’s modified Eagle’s medium (DMEM) (Wako) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin for approximately 2 weeks. Dermal fibrotic cells derived from Dox-inducible β-catenin-expressing mice were supplemented with 0.2 µg/mL Dox.Dermal fibrotic tissues were dissected from the dermal layer of the palmar paws of constitutive active β-catenin-expressing mice (https://www.atcc.org/). They were maintained in DMEM (Wako) supplemented with 10% FBS and 1% penicillin/streptomycin.The RAW264.7 murine macrophage cell line was purchased from American Type Culture Collection  with DMEM supplemented with 10% FBS and 1% penicillin/streptomycin for approximately 2 weeks.n = 7 independent samples) and 10 ng/mL TGF-β1 (Proteintech) for 24 h (n = 5 independent samples). Human Dupuytren’s cells were treated with dimethyl sulfoxide, Wnt inhibitors , 100 ng/mL recombinant Wnt3A (R&D Systems), and 1 ng/mL and 10 ng/mL TGF-β1 (Proteintech) for 24 h (n = 4–7 independent samples). Subsequent analyses, including RNA expression profiling, Western blotting, and immunocytochemistry, were performed on these cells.Murine dermal fibrotic (1st passage) and human Dupuytren’s (1st or 2nd passage) cells were reseeded in 6-, 12-, or 24-well plates for subsequent analyses. Murine dermal fibrotic cells were treated with (0.2 µg/mL) or without Dox for 24 h  supplemented with 60 ng/mL lipopolysaccharide (Sigma–Aldrich) and 40 ng/mL interleukin-4 (PeproTech) for 30 h each.47. For the migration assay, the macrophages were cultured on 12-well plates. At 100% confluence, they were treated with RPMI-1640 medium (Thermo Fisher Scientific) and supplemented with or without 10 nM recombinant mouse CXCL14/BRAK protein (R&D Systems). The bottom of the plates was scratched with a 200 µL micropipette tip and analysed at 0 h and 30 h after scratching.The migration and chemotaxis assay were performed as previously described5 (200 µL) macrophage cells were placed into Costar Transwell chambers on 24-well plates . The bottom wells of the plates were filled with serum-free medium, with or without 10 nM recombinant mouse CXCL14/BRAK protein (R&D Systems). Afterward, the plates were incubated at 37 °C for 30 h. In the chemotaxis assay depicted in Fig. 4 macrophages (200 µL) were seeded into Costar Transwell chambers on 24-well plates. The bottom wells of the plates were filled with 10% FBS containing DMEM (control medium), conditioned medium alone, and conditioned medium with 20 µg/mL of a control IgG  or anti-CXCL14 neutralizing antibody 50. A supernatant culture medium of murine dermal fibrotic cells derived from Tppp3-CreERT2/β-catenin ex3flox mice was used as the conditioned medium. The plates were subsequently incubated at 37 °C for 24 h. The transwell chamber containing macrophages was washed with PBS, and the cells were fixed with 2% paraformaldehyde (PFA) for 20 min and stained with 0.2% crystal violet for 5 min at room temperature. Non-migrating cells on the upper side of the membranes were removed by scraping. The membranes were attached to glass slides. Finally, migrating macrophages were counted using a microscope .For the chemotaxis assay shown in Figs. ACTB/Actb. The primer sequences are listed in Supplementary Table RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN). Up to 1 µg of RNA was used for reverse transcription into cDNA using the PrimeScript RT reagent kit (TAKARA). Real-time quantitative RT-PCR (RT-qPCR) was performed using Premix Ex Taq™  (TAKARA). Transcript levels were analysed with three technical replicates and normalized to those of All murine and human tissue samples were fixed with 4% PFA overnight. The samples were decalcified with pH 7.2 EDTA buffer  for 2 weeks and embedded in paraffin. The samples were sectioned into 3 μm-thick slices. Haematoxylin and eosin (H&E) staining and Masson’s trichrome staining were performed using standard protocols. Primary antibodies for immunohistochemistry included anti-αSMA [1A4] , anti-β-catenin [14/Beta-Catenin] , anti-TPPP3 [GTX33554] , anti-RFP [ARG55744] , anti-CD31 [D8V9E] , anti-CD31 [ab28364] , anti-CD68 [D4B9C] , anti-CD68 [E3O7V] , anti-CD68 [FA-11] , anti-CD206 [E6T5J] , anti-TGF-β [TB21] , anti-CXCL14 [N3C3] , and anti-CXCL14 [MAB730] . The samples were incubated with these primary antibodies at 4 °C overnight.The secondary antibody for 3, 3′-diaminobenzidine staining was Dako EnVision . The samples were incubated at room temperature for 30 min, and the stained cells were analysed using microscopy . For immunofluorescence, the secondary antibodies were conjugated with Alexa Fluor 488 and Alexa Fluor 594 (Thermo Fisher Scientific), followed by incubation with the cells at room temperature for 2 h. Nuclei were counterstained with DAPI . The stained cells were analysed using fluorescence microscopy .Cultured cells were washed with PBS and fixed with 2% PFA for 15 min at room temperature. Next, the cells were treated with a blocking reagent containing 1% bovine serum albumin  for 1 h at room temperature. Antibodies used for immunocytochemistry included anti-αSMA [1A4] , anti-Ki67 [SP6] , anti-Smad2/3 [D7G7] , anti-β-catenin [14/Beta-Catenin] , anti-TGFBR2 [2D5H7] , and anti-CXCL14 [N3C3] . The samples were incubated with these primary antibodies at 4 °C overnight. The secondary antibodies were conjugated with Alexa Fluor 488 and Alexa Fluor 594 (Thermo Fisher Scientific), followed by incubation with the cells at room temperature for 2 h. Nuclei were counterstained with DAPI . The stained cells were analysed using fluorescence microscopy .https://imagej.nih.gov/ij/index.html).Cultured cells were harvested in 150 µL of RIPA lysis buffer, and the protein concentration was measured. Proteins were denatured with 2× SDS at 95 °C for 5 min, and 20 μg of the denatured protein was loaded onto a 10% SDS-PAGE gel. The separated proteins were transferred to a polyvinylidene fluoride membrane . The membranes were treated with a blocking reagent containing 5% BSA (Sigma–Aldrich) for 1 h at room temperature. Primary antibodies were applied in Can Get Signal Solution 1  overnight at 4 °C and secondary antibodies in Can Get Signal Solution 2 (TOYOBO) for 1 h at room temperature. Pierce ECL Plus Western Blotting Substrate (Thermo Fisher Scientific) was used for visualization. Blots were scanned with ImageQuant™ LAS4000 mini4000 (Cytiva) and quantified using ImageJ , anti-αSMA [1A4] , anti-β-catenin [14/Beta-Catenin] , anti-LaminB1 [12987-1-AP] , anti-CXCL14 [N3C3] , and anti-β-actin [13E5] ; the secondary antibodies used were HRP-linked anti-rabbit IgG [#7074] and anti-mouse IgG [#7076] antibodies .Tppp3-CreERT2/β-catenin ex3flox mice were treated with 1 mg of tamoxifen for three consecutive days and were administered 15 µg  of a mouse monoclonal anti-CXCL14 neutralizing antibody [MAB730] (R&D systems) or 15 µg  of mouse monoclonal control IgG [C1.18.4] (Bio X Cell) twice a week for 1 month. All mice were euthanized by cervical dislocation at 2 months of age, and the forepaws were collected.One-month-old t-test or non-parametrical two-tailed Mann–Whitney U test was used. For the comparison of the Ki67- and αSMA-positive cell ratio of murine dermal fibrotic cells, αSMA-positive cell ratio of human Dupuytren’s contracture-derived cells, migration and chemotaxis assays of RAW264.7 cells, dermal thickness, and number of myofibroblasts and CD206-positive mouse cells in immunocytochemistry, a non-parametrical two-tailed Mann–Whitney U test or one-way analysis of variance (ANOVA) with the Tukey or Tukey–Kramer multiple comparison test was used. Statistical analyses were performed using GraphPad Prism 9.4.0. Differences were considered statistically significant at p < 0.05.Data from the RT-qPCR, immunocytochemistry, Masson’s trichrome staining, migration assay, chemotaxis assay, and immunohistochemistry assay are presented as mean ± standard deviation (SD). For the statistical comparison of RT-qPCR data, a parametrical two-tailed paired Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupporting Data 1Reporting Summary


The aim of this research was a comparative analysis of selected mechanical properties of epoxy compounds that were modified with metallic fillers and aged in aqueous environments. The tested epoxy compounds consisted of three components: styrene modified epoxy resin based on Bisphenol A, triethylenetetramine curing agent (resin/curing agent ratio of 100:10) and two types of metallic fillers in the form of particles: aluminum alloy (EN AW-2024–AlCu4Mg1) and tin-phosphor bronze (CuSn10P). Samples were subjected to ageing in 4 water environments: low-, medium- and high-mineralized natural water and in a sugar-containing solution for 1, 2 and 3 months. The epoxy samples were subjected to compressive strength tests in accordance with the ISO 604:2002 standard. It was observed that, among others, the compositions seasoned in low-mineralized water usually achieved the highest average compressive strength. As for filler type, using the bronze filler (CuSn10P) usually achieved the highest average compressive strength results. Modifications of polymeric materials are aimed at improving selected properties of the cured material . They arFor polymer materials, there are several types of modifications: chemical modifications, physical modifications and physical–chemical modifications. Chemical modifications involve, for example, changing the type and amount of hardener to adjust the curing time, strength, chemical and low/high temperature resistance of the material ,19,20,21Adding reduced graphene oxide (RGO) up to 0.5 wt% increased the glass transition temperature, the modulus of elasticity and the strength of the epoxy adhesive and reduced the adhesive’s strain at the break at the ambient temperature, but the ultimate strain of RGO-reinforced adhesives decreased slightly . VariousAluminum oxide, silica and asbestos (inorganic fillers) increase strength and also contribute to heat resistance ,34,35. FHowever, the use of modifiers can lead to undesirable effects. The most important of these is increased viscosity of the composition, which makes it more difficult to process. In addition, problems such as poor adhesion, blistering, unevenness or discoloration on the surface and other potentially unfavorable hazards can occur.The operating environment of polymeric materials has a significant impact on their strength and durability. Aging is associated with changes in the characteristics of the polymer material due to factors such as UV radiation ,37,38, tMany construction solutions require materials with significant compression strength and adhesive properties, as they are intended as joining or sealing materials . In manyIn this paper, a comparative analysis of selected mechanical properties of epoxy compounds modified with metallic fillers  that were seasoned in aqueous environments was carried out. Five experimental environments were analyzed as aqueous environments: three aqueous environments with different degrees of mineralization , a carbonated beverage with high sugar content and an ambient environment in which the seasoning process was not implemented—for comparison purposes. The experimental study provided information on the properties of modified epoxy compounds exposed to aqueous environments, which can be used in the design of adhesive joints and the development of adhesive technology.The following components were used in epoxy compounds: a styrene-modified epoxy resin based on a bisphenol A (Epidian 53) with an epoxide number of min. 0.41 mol/100 g (DGEBA and styrene) and a triethylenetetramine curing agent (Z-1 trade name) with an amine number of min. 1100 mg KOH/g. Both main components of the epoxy composition were produced by the “Organika-Sarzyna” Chemical Plant in Nowa Sarzyna, Poland.The selected properties of the epoxy resin and curing agent are shown in A triethylenetetramine curing agent (amine type) used most often for compounds with low molecular weight epoxy resins. Two types of particulate materials were used as metallic fillers: aluminum alloy (EN AW-2024 AlCu4Mg1) and tin-phosphorus bronze (CuSn10P).Aluminum alloy (EN AW-2024) is a lightweight metal with low oxidation resistance and is difficult to weld. It is characterized by a large amount of copper and high fatigue strength. These materials find important applications in the automotive and aerospace industries, as well as in the construction industry—if there is no risk of corrosion. The physical properties of the aluminum alloy used are shown in Tin-phosphorus bronze (CuSn10P) is an alloy of copper with tin and an admixture of phosphorus. The copper content is approximately 90%, the tin content is 1–9% and the phosphorus content is 0.1–0.3%. It is typically used in the manufacture of components for heavy-duty and high-speed operation, e.g., corrosion-prone bearings; machine parts including shafts, bushings and other components; and chemical fittings. Since tin-phosphorus bronze is characterized by very good self-lubricating properties, it is used for the manufacture of poorly lubricated components, such as those that are difficult to access and exposed to long intervals of operation without maintenance. The particle size of the tin-phosphorus bronze was 0.05 ÷ 0.10 mm. The basic physical properties of bronze are shown in The main components of the epoxy composition, the resin and curing agent were mixed, taking into account the appropriate stoichiometric ratio recommended for these specific components. For this resin and curing agent, the stoichiometric resin:curing agent ratio is 100:10. The compounds adopted in the research plan  were preThe following conditions were used during curing: temperature: 20 ± 2 °C, humidity: 26 ± 1% and curing time: 7 days. A total of 78 samples of each epoxy compound were made.Five test environments were used for the study. The aqueous environments encompassed water classified into three different groups according to the current Regulation of the Polish Minister of Health of 31 March 2011 on natural mineral waters, spring waters and table waters, Annex 5—“Criteria for chemical classification used in the labelling of natural mineral waters”. These were: low-mineralized water, medium-mineralized water and high-mineralized water . A carboThe water types used for testing are extracted from underground and show purity and the absence of chemical as well as bacterial contamination. They all have a one-year expiry date set by the manufacturers  and specific storage conditions (dark and cool places). The ingredients of the sweet drink used in the study are water, glucose-fructose syrup, sugar, color, ammonia-sulphite caramel, phosphoric acid, acidity regulator and flavors. It is also unlike the waters presented above in being carbonated. This drink, according to the manufacturer’s recommendation, should be stored in shaded and cool areas.The modified epoxy samples were cured for 7 days at 20 ± 2 °C and humidity: 26 ± 1%. Series of 6 cured samples were placed in varying operating environments including one series in ambient conditions. The seasoning times for each series were: 1, 2 and 3 months. The seasoning conditions  were identical for all samples.After fabrication, the specimens of the cured epoxy compounds were milled to remove surface irregularities, which was necessary for proper strength testing. Compressive strength tests were carried out on a ZWICK/ROELL Z150 machine  according to the ISO 604:2002 Plastics—Determination of compressive properties , with coThe coefficient of variation (COV), as a measure of the variability of the data in a series of samples, describes the degree of dispersion or variability of the data (standard deviation SD) in relation to its mean value (MV). It is expressed by the Formula (1):A coefficient of variation of less than 15% is estimated to define a series with low variability, i.e., those in which the individual values are relatively close to the mean value and there are no large discrepancies between them.It can be observed that all the series studied had a very low variability, somewhere between 1 and 9%. At the same time, there is a clear difference between the variability of the results of the individual series depending on the tested epoxy material. The E53/Z-1/Al showed almost twice the average variability of the unmodified epoxy compound (E53/Z-1), while E53/Z-1/Br showed more than twice as much variability. Still, the results of the dispersions make it possible to describe all series as having low variability. This indicates, on the one hand, a high consistency of results and, indirectly, also a correctly selected minimum number of samples in each series.A more detailed analysis of the dependence of the degree of dispersion of the results in relation to the seasoning time and the seasoning environment did not show similar relationships.The results of the unseasoned epoxy compound samples that were not seasoned in aqueous environments were analyzed. The samples with the epoxy compound Epidian 53 with Z-1 curing agent and bronze filler (E53/Z-1/Br) clearly showed the highest average compressive strength of 83.32 MPa. The difference between the average strength of the strongest and weakest samples was only 5%. The scatter in the results obtained with the E53/Z-1/Al epoxy compound was noticeably greater than with the other two epoxy compounds.2-saturated high-sugar beverage, the differences were only about half the size.When analyzing the average results obtained after 1 month of seasoning , the folThe individual compositions responded differently to the seasoning environment. E53/Z-1 between the highest  and lowest average strength (for the high-sugar beverage 79.90 MPa) achieved a difference of 4.78 MPa (5.65%). For the E53/Z-1/Al composition, the difference was 3.73%  and E53/Z-1/Br 5.93 MPa, i.e., 6.75% .Looking in detail at the results obtained from the samples after 2 months of seasoning, it can be seen that the highest average compressive strength among the compositions tested was obtained for samples seasoned in highly mineralized water in the case of the E53/Z-1/Br composition (86.08 MPa), while the lowest was obtained for the E53/Z-1 composition seasoned in a high-sugar beverage (77.6 MPa). The difference between these extreme averages was 8.48 MPa, which represents 9.8%. The behavior of the tested compositions in low-mineralized water and the high-sugar beverage was quite similar, as can be seen by only a 3.2% and 4.23% difference between the largest and smallest results obtained. However, in the case of the high-mineralized water, it was 6.75%, and in the medium-mineralized water, it was 8.3% (78.72 MPa–E53/Z-1/Al vs. 85.84 MPa E53/Z-1/Br).Analyzing the results obtained after 3 months of seasoning, the following observations can be noted. There is a clear difference of up to 16.5% between the most and least durable composition . Within the individual seasoning environments, the differences were significantly smaller: 8.3% in the low-mineralized water, 6.6% in the high-sugar beverage, 5.4% in the medium-mineralized water and 2.6% in the high-mineralized water.An attempt to summarize all the results obtained for the compressive strength of the epoxy material samples grouped according to the type of environment and seasoning time is shown in Analyzing the results obtained after seasoning in low-mineralized water, it was noted that, irrespective of the seasoning time, the difference between the highest (E53/Z-1/Br for 3 months: 90.58 MPa) and lowest (E53/Z-1/Al for 1 month: 82.37 MPa) average compressive strength obtained was 8.21 MPa (9.1%). Within the individual epoxy compounds, in fact, a notable difference was only observed between the samples made from E53/Z-1/Br seasoned for 1 and 3 months: namely, the 3-month samples proved to be 6.3 MPa stronger (7%). The other two compositions had differences between minimum and maximum average compressive strengths in the range of 2–3%.In the case of medium mineralized water, the higher average strength of the epoxy samples filled with tin-phosphorus bronze was clearly seen, especially at the beginning of the seasoning. The difference between the least durable material (E53/Z-1/Al seasoned 2 months—78.72 MPa) and the most durable (E53/Z-1/Br seasoned 1 month—87.65 MPa) exceeded 10% (8.93 MPa). Comparing the average values obtained for the different materials, it is evident that the influence of the seasoning of the undoped material (E53/Z-1) and aluminum-doped material (E53/Z-1/Al) is very small: 0.7–1.2%. Only the samples in which bronze (E53/Z-1/Br) was used achieved a decrease in average compressive strength of 4.2 MPa (approx. 5%).Samples aged in highly mineralized water showed average strength changes with seasoning time. Globally, a 6.7% difference was registered between the strongest and weakest samples—the average strength of the bronze-doped epoxy was 5.8 MPa higher than that of the aluminum-filled epoxy. It was the E53/Z-1/Al samples that were the most stable during seasoning, showing only a 1.5% difference between the seasoning series. A 4.1–4.5% difference (3.55 MPa–3.81 MPa) was observed between the undoped and bronze-doped samples.2-saturated, high-sugar beverage. These values were essentially unchanged over time (0.1% difference). A slightly larger change was registered for epoxy with bronze (E53/Z-1/Br)—2.4%. In contrast, for the unmodified specimens, degradation progressing with seasoning was observed, changing by 4.3 MPa (5.3%). Comparing all materials with each other, the greatest difference was observed between the unmodified samples (75.63 MPa at month 3 of seasoning) and the modified bronze, which reached an average strength of 91.72 MPa after one month of seasoning. This gives a 7.5% difference between the averages.Using aluminum filler again resulted in the greatest stability of the average compressive strength results when the material was seasoned in a COThe results of the compressive strength tests were analyzed according to the type of epoxy compound—E53/Z-1, which was seasoned in four environments for one, two and three months, and epoxy compounds that were not seasoned. The samples that were not seasoned achieved an average compressive strength of 79.24 MPa. The seasoned epoxy compound showed little change in average strength in two cases: when seasoned in low- and medium-mineralized water. In highly mineralized water and the Cola beverage, a progressive decrease in average compressive strength of up to approximately 5% was recorded with seasoning time. When analyzing the differences between the various compositional behaviors in the seasoning environments studied, it is worth noting that the epoxy compound showed a particularly large deterioration in compressive strength during seasoning in the carbonated high-sugar beverage, with the average values in this group being noticeably lower than in the mineralized water groups.The aluminum-doped composition, as noted above, proved to be quite stable and resistant to seasoning in the analyzed environments. In case of low-mineralized water—the difference in average compressive strength during seasoning did not exceed 3.6%. Water with a higher mineral content caused even less of a change in average strength: medium—0.5%, high-mineralized—1.5%. Similarly, a carbonated beverage with high sugar content registered a 0.2% change. Regarding the overall effect of the seasoning environment on the average compressive strength value, it is clear that lower values were obtained in the medium mineralized water compared to the other waters and the beverage. Meanwhile, the highest values were obtained in the group of samples seasoned in low mineralized water. However, it is difficult to draw detailed conclusions on this basis, as precisely these two groups  were characterized by clearly higher standard deviation values.Analyzing the results obtained, it can be seen that quite large differences were registered within the samples seasoned in different environments. Between the least and most durable variants, the difference reached 12%. Clearly, the lowest values of average compressive strength were obtained with the epoxy compound in the environment of a carbonated beverage with high sugar content. And in this environment, the results obtained were the most consistent, with a min-max difference of 2.4%. The less mineralized the water used for seasoning, the greater the differences within the group. That is, for highly mineralized water, 3.5 MPa (4.1%) was recorded between the lowest and highest mean strength values, for medium mineralized water it was 4.2 MPa (4.8%) and low mineralized water showed a difference of 6.3 MPa (7%).The studied epoxy compounds were based on Epidian 53 epoxy resin, Z-1 curing agent and fillers in the form of particles of aluminum alloy (EN AW-2024) and tin-phosphorus bronze (CuSn10P). The cured compounds were seasoned in four experimental environments—low-, medium-, and high-mineralized water and also in a carbonated high-sugar beverage for periods of one, two and three months.The application of fillers to polymeric materials, in this case epoxy, was carried out in order to obtain better properties of the cross-linked material. It may happen that for the price of improving selected characteristics, others may deteriorate. The selection of the right filler and its amount for use in the epoxy is therefore crucial to achieving the desired goals. Hence, experimental studies on the effect of selected fillers on the various characteristics of specific epoxy compounds are needed.Epoxy materials can be degraded by water sorption. The mechanism of this interaction has been analyzed in many works. When testing the effect of water on epoxy coatings, using infrared spectroscopy and dielectric measurements, the appearance of hydrogen bonds was observed during hydrothermal aging . The anaThe degree of saline water absorption by epoxy coatings and the swelling of these coatings, regardless of the solution temperature, were lower for coatings with physical aging than for coatings without aging. Thermodynamic analysis of the diffusion parameters showed that thickening of the polymer matrix induced by physical aging and, consequently, a higher number of interactions between the polar groups of polymer chains, which reduce the number of polar groups for water diffusion, are the main factors regulating the process of water absorption . SimilarThe progress of the crosslinking process is also important. It has been shown that depending on the curing temperature of DGEBA/TETA compounds, despite the achievement of complete crosslinking, different curing conditions affect the free volume and activation volume associated with viscoelastic properties. Compounds cured at temperatures below Tg exhibit higher mechanical properties and are less susceptible to water ingress than systems cured at temperatures above Tg .In the present research, degradation of the average compressive strength of epoxy compound samples with metallic fillers was recorded in almost all of the analyzed cases of aging the epoxy material in a mineral water environment. Only the epoxy compound filled with aluminum resisted deterioration of properties when it was seasoned in medium and highly mineralized water. On the other hand, ageing in the environment of a carbonated beverage with high sugar content did not lead to such a clear decrease in the compressive strength properties.To conclude, when ageing the epoxy compounds in low-mineralized water, the results show a slight improvement in strength properties. This may be due to the presence of fewer mineral components. These components generally have a negative effect on the strength of epoxy compounds.The right choice of filler type affects the mechanical strength of the epoxy material. The study showed that bronze was the most favorable filler. The results may have indicated a higher strength due to a better distribution of bronze particles in the cylindrical molds used to shape the samples from the tested composition.The topic of modification of epoxy compounds with metallic fillers is extensive and needs to be further explored. Conducting further research will allow us to continuously improve and gain more knowledge about the modification of epoxy compounds with metallic fillers.The epoxy compound that has been seasoned in low-mineralized water usually obtains the highest strength results: the highest average compressive strength was achieved by E53/Z-1/Br samples, which were seasoned in low-mineralized water for a period of three months; the strength of this epoxy compound is 90.58 MPa,The seasoning environment—in the form of a carbonated beverage—is not particularly beneficial to obtaining high-strength results; it was during the seasoning of the epoxy compound in this environment that the lowest strength results usually occurred,The epoxy compound with bronze filler (CuSn10P) tended to obtain the highest compressive strength results,Samples of epoxy compounds without filler usually achieved the lowest strength results: the lowest average strength was achieved by the E53/Z-1 epoxy compound, which was seasoned for a period of three months in a carbonated high-sugar beverage and reached an average of only 75.63 MPa.This study was conducted to test the compressive strength of epoxy compounds with metallic fillers. Analysis of the results established that the environment and seasoning time affect the compressive strength results. The following conclusions can be drawn:In general, it can be seen that the content of minerals in the aqueous environment influences the mechanical properties of epoxy materials, and a smaller amount of minerals is recommended,A sugar-containing environment negatively affects the properties of epoxy materials; therefore, exposing such materials to this type of environment should be avoided,During modification, the type of modifying agent is important, which is why it is necessary to analyze the use of epoxy materials in specific construction solutions along with the required strength properties.The practical recommendations resulting from the research include: