The aim of this work is to assess the influence of different commercial diluents on some mechanical properties of two bisphenolic epoxy compounds, cold-cured by a polyamide curing agent, to be employed as epoxy structural adhesives for building and industrial applications. The diluents under analysis were epoxy, bituminous, nitro, acrylic and extraction. The choice of these products was made on the basis of their wide commercial availability as diluents for epoxies used as adhesives and in different industrial and construction applications. The diluents were all added in small proportions, i.e., from 1 to 10 g per 100 g of epoxy resin. The cold-cured epoxy compounds were subjected to compressive (according to ISO 604) and static tensile (according to ISO 527-1) tests. The same mechanical tests were performed on both unmodified epoxy resins, for comparison purposes. On the basis of the obtained results, it was concluded that the influence of the presence of a diluent, and of its amount, on the mechanical properties of epoxy compounds depends on the type of resin and of diluent, as well as on the mechanical characteristics analyzed. Epoxy resins are currently widely employed in many different industrial sectors because of their ease of processing and curing, even at different temperatures, and extremely good mechanical properties ,2,3,4. TEpoxy resins can be modified to obtain specific final characteristics: this can be done by adding proper modifiers depending on the property to be achieved ,23. In tHighly viscous epoxy resins are difficult to process; this can represent an obstacle during their transformation to produce different components also containing fibers. To overcome this limitation, diluents can be introduced. The main characteristics of a diluent or a viscosity modifier for epoxy resins were presented by Monte  and JagtNon-reactive diluents are most commonly used since they do not affect the kinetics of the curing reaction of the resin . Non-reaLow-viscosity epoxy resins can be used to dilute diane-based epoxy resins, affecting their curing kinetics. Non-reactive diluents, on the other hand, do not generally reduce the reactivity of epoxies and can be, therefore, added in large amounts. Epoxy compounds containing diluents can be cold- or heat-cured; the properties of the obtained cured systems depend also on the type and amount of diluent added ,38,43. OEven highly polar non-reactive diluents can affect the properties of cured epoxies; they are also known as anti-plasticizers. For example, upon the addition of 7–11 pph (parts per hundred) of a non-reactive diluent  in a bisphenolic epoxy resin, an increase in its modulus of elasticity, hardness, tensile, flexural and compressive strength was recorded, but also reductions in elongation at break and impact strength.Cured epoxy compounds containing different diluents were investigated in the present study. Two bisphenolic epoxy resins were selected, i.e., a standard epoxy and an epoxy modified with styrene. They were both cold-cured with a polyamide curing agent. Five different diluents  were added to both resins in three amounts . The selected diluents are the most widely used to produce epoxy structural adhesives for building and industrial applications; they are also widely commercially available. The epoxy compounds, cured at ambient temperature, were subjected to compressive and static tensile tests to assess the effects of each diluent on the mechanical properties of both cured epoxies. The findings of the present study will be also of invaluable utility for the processing and industrial applications of such materials.Thirty variants of cured systems, based on two epoxy resins modified with different diluents, were produced and analyzed. Two types of epoxy resins were employed: a standard bisphenolic epoxy resin, with the trade name Epidian 5, displaying an epoxy number of 0.48–0.51 mol/100 g, and a modified (with styrene) epoxy resin, with the trade name Epidian 53, with a minimum epoxy number of 0.41 mol/100 g. Both epoxy resins were supplied by Sarzyna Resins .3 (data reported on datasheet of Epidian 5).Epidian 5 is an epoxy resin based on bisphenol A, which is the base resin widely used as an adhesive and matrix of composites for various applications. This epoxy displays a maximum viscosity of 3000 m·Pas (at 25 °C) and density at 20 °C of 1.17 g/cm3, measured at 20 °C. Epidian 53 resin has an average molecular weight of less than 700. The previous data was taken from the manufacturer’s resin data sheet.Epidian 53 consists of a mixture of bisphenol A and epichlorohydrin. It is a styrene-modified epoxy resin (the amount of styrene ranging from 13 to 15 g per 100 g of resin). This resin has a lower viscosity (ranging from 900 to 1500 m·Pas at 25 °C) and a density of 1.11–1.15 g/cmBoth resin systems were cold-cured upon the addition of a polyamide curing agent, indicated as PF . This curing agent is characterized by an amine number of 350–400 mg KOH/g. Epoxy compounds were produced by mixing each epoxy resin with the PF curing agent in the ratio 100/50, as indicated by the supplier. The main properties and characteristics of the materials used in this study, i.e., the two epoxy resins and the polyamide curing agent, can be found in ,44,45.The epoxy compounds  are widely used in the production of adhesives to join different materials. The addition of a suitable diluent in the adhesive mix may bring some advantages, both technological  and in terms of final performance.In this work, the term “epoxy compound” indicates the mixture consisting of one of the selected epoxy resins and the hardener PF, with the possible addition of a diluent.Five diluents were used in the investigation, namely epoxy, bituminous, nitro, acrylic and extraction. They were all provided by Dragon Poland Company . All diluents are reported as low-viscosity substances, even though the exact values of their viscosity are not reported on their datasheet. The diluents reported in The epoxy diluent can be effectively employed as diluent for different epoxy products because of a similar chemical nature. It has a low sulfur content and it does not contain benzene; in addition, it does not change the color of the diluted resin. The bituminous diluent is a mixture of hydrocarbons containing ethylbenzene, xylene, ethyl acetate and butyl acetate, while the nitro one is a mixture of ethanol and ethyl acetate. This latter has a low sulfur content and contains additives able to improve the flow (decreasing the viscosity) of epoxy paints and coatings; its addition does not lead to yellowing of the diluted resin. The acrylic diluent contains C8–C10 alkyl polyglucoside and ethyl acetate, with a low sulfur content. Finally, the extraction diluent contains low amounts of enzymes in hydrogen and hexane. Further information on the diluents used can be obtained from the technical data sheets of the materials, provided by the manufacturer of all products .Each diluent was added in one of the two epoxy resin in three amounts, i.e., 1 g, 5 g and 10 g per 100 g of epoxy resin. Therefore, three variants for each couple of epoxy resin/diluent were produced, with a total number of 30 systems, whose compositions and designations are reported in Samples of the epoxy compounds suitable for compression and tensile tests according to ISO 604 and ISO 527-1 standards, respectively, were produced.The resin, curing agent and diluent of each epoxy compound, in the amounts reported in The specimens for compressive tests were fabricated in cylindrical shape, as illustrated in For the tensile tests, six dog-bone samples for each compound, according to the compositions reported in All the specimens were produced by casting the liquid mixtures in molds of proper shape and dimensions: the cylindrical samples were fabricated employing polyethylene molds of 10 mL capacity while the dog-bone specimens were prepared in silicone molds. All the specimens were cured at 22 °C and 22 (±1)% R.H. for 14 days. After the curing process, the specimens were milled  to obtain flat and perpendicular surfaces to assure a correct clamping of the specimens in the grip on the testing machine. The milling was performed with a FNB26-type milling machine . Mechanical tests were performed on the produced specimens with the aid of a Zwick/Roell Z150 testing machine . From the results of such experiments, it was possible to compare the mechanical behavior of the epoxy compounds, assessing the effects of the different solvents added in diverse amounts.M (MPa).Cylindrical samples  were subt (MPa) were recorded during the tests.The mechanical tests in tensile mode were performed according to ISO 527-1 standard on dog-bone specimens, shown in The effect of the selected diluents, added in different amounts, on the mechanical characteristics on the two epoxy resins under investigation, i.e., Epidian 5 and Epidian 53, was first assessed by performing the tests in compression mode. The compressive modulus values measured on epoxy compounds upon the addition of epoxy diluent are illustrated in The highest value of compressive modulus was observed for the reference Epidian 5 bisphenolic epoxy resin, i.e., the EP5/PF system. The epoxy resin modified with styrene, i.e., Epidian 53, displayed an appreciably lower compressive modulus, probably because of the lower epoxy number of this latter resin, and mainly to its modification with styrene. When the epoxy diluent was added in Epidian 5 resin, an appreciable decrease  in compressive modulus was recorded, particularly at greater (5 g and 10 g) amounts. The data obtained for Epidian 53 resin with the epoxy diluent are generally lower than the values measured on formulations prepared with Epidian 5 epoxy, with the exception of EP53/PF system containing the largest amount of this diluent. Excluding this latter, the effect of the epoxy diluent is again revealed in a steady reduction in the compressive modulus by increasing the amount of the diluent. An unexpected behavior was observed for EP53/PF/REP 100:50:10 system, with a value of compressive modulus even greater than that measured for the unmodified resin. The standard deviation calculated for the results of these tests was in all cases not greater than 10–11%, testifying to a good reproducibility of results.The addition of this diluent affects in different ways the compressive modulus of the two investigated resins. It produces an appreciable decrease in this property in the case of Epidian 5 epoxy resin, even at very low amounts (1 g and 5 g) of diluent. The influence of the diluent content, in fact, is almost negligible for this resin. On the other hand, the higher compressive moduli were obtained for EP53/PF/RB epoxy compounds at the lower contents of bituminous diluent, i.e., 1 g and 5 g. These values are even greater than that measured on unmodified Epidian 53 epoxy. When adding a greater amount of bituminous diluent to this resin, on the other hand, a sharp drop in compressive modulus is recorded, which falls to a value similar to that recorded for the other resin containing the same amount of the diluent. In addition, in these tests, a standard deviation never exceeding 10% was calculated. When nitro diluent was added in both epoxy resins, the compressive modulus values reported in A different trend was observed for the compressive modulus measured on Epidian 5 and Epidian 53 resins, respectively, containing nitro diluent in increased amounts. A significant decrease in this property  was recorded for Epidian 5 epoxy at the lowest content of nitro diluent. At greater diluent contents, a slightly greater value of compressive modulus was recorded that, however, remains well below that measured on Epidian 5 epoxy. When the same diluent in the lowest amount (1 g) was added to Epidian 53 epoxy resin, a compressive modulus 25% greater than that measured on the pristine resin was measured. Conversely, by increasing the content of nitro diluent, an appreciable drop in this property was recorded, being the differences between 5 g and 10 g contents almost negligible, taking into account the standard deviation of results.The moduli calculated in compressive mechanical tests performed on epoxy compounds, unmodified and modified with acrylic diluent, are presented in Finally, in From this attentive analysis, it was concluded that the effect of the addition of the different diluents on compressive modulus of the epoxy compounds, based on two epoxy resins, depended on the kind of resin and on the type of diluent added to each epoxy. Generally speaking, the diluents had a detrimental effect on this property when analyzing Epidian 5 resin, i.e., a standard bisphenol A epoxy resin, characterized by a higher value of compressive modulus in its unmodified formulation. The only exceptions were represented by the acrylic and extraction diluents, whose addition in moderate amounts led to enhanced compressive modulus. Referring to the styrene-modified epoxy, i.e., Epidian 53, the diverse diluents differently affected its compressive modulus without a clear trend. It seemed, moreover, that the diluents were able to increase to a certain extent (depending on the kind of diluent) the compressive modulus of this resin but only at the lowest diluent content, with the exception of epoxy diluent.The mechanical tests, carried out in compression mode on Epidian 5 and Epidian 53 epoxies modified by the different diluents, allowed us to assess the influence of these modifiers on the compressive strength of both resins. Similar values of the compressive strength were measured for both epoxies, i.e., the Epidian 5 and Epidian 53 resins. The effect of the epoxy diluent was more evident for the Epidian 53 epoxy, leading to generally improved values of its strength. No clear trend can be attributed to the content of the diluent in this resin: the strength is almost four times greater than that found on the unmodified resin at the lowest (1 g) diluent content; it is nearly doubled at 5 g of diluent and it rises again (+150%) at the greatest selected content. More limited was the growth in compressive strength measured on Epidian 53 resin upon the addition of the epoxy diluent—the greater the amount of solvent, the lower the compressive strength. In addition, in the case of the compressive strength data, the standard deviation of results never exceeded 10%. M achieving the greatest value at the lowest modifier content. A general decrease in compressive strength was observed as the amount of bituminous diluent increased, irrespective of the resin type.In addition, in the case of bituminous diluent, its addition leads to an increase in compressive strength compared to the respective unmodified resins, i.e., Epidian 5 and Epidian 53. Again, the growth in strength was more marked for Epidian 5 resin, σThe comparison of the strength data obtained from mechanical compressive tests performed on epoxy compounds, with or without nitro diluent, is shown in The compressive strengths recorded on compounds based on both epoxy resins, i.e., Epidian 5 and Epidian 53, were in line with what was previously observed: a substantial increase in compressive strength upon the addition of the diluent, more significant for Epidian 5 epoxy. This increase was generally reduced by raising the diluent content, with the exception of systems based on Epidian 53 epoxy. In this epoxy, in fact, the highest value of compressive strength was calculated when 5 g of nitro diluent were added.The trend observed for the compression strength values that were due to the addition of acrylic diluent in different amounts was substantially similar to what already observed for the other modifiers. It is confirmed, therefore, that the addition of a diluent modifier brings about an increase in compressive strength to both epoxies, and in particular to Epidian 5 epoxy resin, this growth being reduced at increased diluent contents.Similar conclusions can be drawn by the observation of the results, in terms of strength, of the compressive tests performed on the epoxy compounds unmodified and modified with extraction diluent, as shown in Accordingly, the extraction diluent produced a noticeable increase in the compressive strength of Epidian 5 resin; by increasing the amount of this diluent, the strength value declined, although still remaining well above the value measured for pristine Epidian 5. Similarly, the compounds based on Epidian 53 epoxy experienced a growth in the compressive strength upon the addition of the extraction diluent. The lowest increase (around 45%) was in this case observed when 5 g of diluent were added.In conclusion, it is possible to attribute a positive role to all diluents analyzed, all being able to improve, even if to different extents, the compressive strength of both epoxy resins under investigation. Generally speaking, the best enhancements can be achieved for the bisphenolic Epidian 5 resin at the lowest (1 g) diluent content, this last occurrence representing an advantage from an operative point of view. At this content, Epidian 5 epoxy resin achieved compressive strengths four times greater than the strength measured on the unmodified resin, i.e., about 117 MPa. Epidian 53 epoxy, on the other hand, displayed the highest compressive strength value of about 76 MPa upon addition of 5 g of nitro diluent.The investigation continued, then, to analyze the effects of the chosen diluents on the tensile mechanical properties of Epidian 5 and Epidian 53 epoxy resins. The results of the performed tests in terms of tensile modulus are first presented. In Comparing first the tensile modulus of the unmodified resins, it can be noticed that Epidian 5 epoxy resin offers a greater modulus with respect to Epidian 53, even testing both materials in tensile mode. This result can be again justified by the lower epoxy number of Epidian 53 resin, leading to a lower cross-linking density with respect to Epidian 5 epoxy and, in turn, to a lower stiffness. The modification of a bisphenolic epoxy resin with styrene, thus producing Epidian 53 resin, may also have contributed to the lower tensile modulus measured for this resin. Nevertheless, a very high stiffness value results in an excessive fragility of the epoxy, which is unsuitable for some applications. The data shown in The tensile moduli calculated on epoxy compounds based on Epidian 5 and Epidian 53 resins, unmodified and modified with bituminous diluent, are presented in In addition, the bituminous diluent was able to improve the tensile modulus of both resins, irrespective of its content in the mixtures. In the case of Epidian 5 epoxy compounds, the greater the content of the bituminous diluent, the higher the tensile modulus value, at least in the range of compositions analyzed. Conversely, the formulations based on Epidian 53 resin displayed values of tensile modulus that decreased by increasing the amount of diluent, still remaining greater than the same characteristic measured on the pristine resin. The tensile modulus values recorded on Epidian 5 and Epidian 53 epoxy compounds, unmodified and modified with nitro diluent, are shown in As for the previously analyzed diluents, a general increase in the tensile modulus was recorded upon the addition of nitro diluent to Epidian 5 and Epidian 53 resins. The addition of greater amounts of nitro diluent caused a gradual decrease in the tensile modulus measured on compounds based on Epidian 53 resin. The higher values of this characteristic were obtained in compounds based on Epidian 5 epoxy containing 1 g and 5 g of nitro solvent. Beyond this composition, the tensile modulus of such systems experienced a strong reduction, nonetheless remaining greater (+11%) than that of the unmodified Epidian 5 resin.The addition of acrylic diluent produced an increase in tensile modulus, irrespective of the kind of epoxy resin. A basically constant decline in tensile modulus was recorded by increasing the amount of this diluent, from 1 g to 10 g, in both resins. The epoxy compounds based on Epidian 5 resin always displayed much greater values of tensile modulus with respect to those calculated on systems based on Epidian 53 epoxy. Standard deviation values were, also in this case, smaller than 10%. Finally, in From the analysis and comparison of the modulus data calculated in the tensile tests performed on the different systems, the following conclusions can be drawn: (i) the addition of diluents in compounds based on both Epidian 5 and Epidian 53 epoxies brought about general improvements in their tensile modulus, irrespective of the epoxy resin and the kind of diluent; (ii) albeit with some exceptions, the greatest modulus values were obtained at low quantities (1–5 g) of each diluent; and (iii) taking into account a few exceptions, the compounds based on the stiffer resin, i.e., Epidian 5 epoxy, displayed generally higher values of tensile modulus than those based on the less stiff Epidian 53 epoxy, irrespective of the type and content of the diluent.The mechanical tests performed in tensile mode allowed us to also measure the tensile strength values as a function of the kind and the amount of diluents added to the Epidian 5 and Epidian 53 epoxies. In The examination of the results of tensile tests revealed that Epidian 53 epoxy resin offers a greater tensile strength (+45%) with respect to Epidian 5 epoxy resin. The value of the tensile strength of this latter, i.e., a bisphenolic epoxy resin cured at 22 °C, is in line with the tensile strength reported for a two-part system composed by a bisphenolic epoxy resin cold cured by a polyamide, i.e., 12 MPa .While continuing to analyze the effects of the epoxy diluent on the tensile strength of the compounds based on the two epoxy resins, it was noticed that this diluent produced an increase in this characteristic, irrespective of the kind of epoxy. The rise in strength was greater at lower diluent contents  for both resins; this characteristic was reduced by increasing the amount of diluent in both epoxy compounds. At a content of 5 g of diluent, the strength measured on Epidian 5 epoxy resin exceeds that calculated on Epidian 53. A standard deviation not greater than 15% testified a good reproducibility of results obtained from these experiments.The diagram reported in A clear trend was observed in this case, i.e., a growth in tensile strength compared to unmodified epoxy resins, which was reduced by increasing the diluent content. At the lowest amount of bituminous diluent, i.e., 1 g, very similar tensile strength values were measured on both epoxy resins. At greater diluent contents, the compounds based on the more resistant resin, i.e., Epidian 53, displayed higher tensile strength with respect to the epoxy systems based on Epidian 5 resin.The comparison between tensile strength values calculated on epoxy compounds based on Epidian 5 and Epidian 53 epoxies, unmodified and modified with nitro diluent, is shown in The highest tensile strength values were found when 5 g nitro diluent were added to both resins; at this concentration, the compound based on Epidian 5 epoxy resin was able to display a greater strength over that based on the more resistant Epidian 53. The tensile strength declined at the lowest (1 g) and greatest (10 g) amounts of nitro diluent, remaining for both epoxies above the values measured on the pristine resins.The results, in terms of strength, of the tensile mechanical tests performed on Epidian 5 and Epidian 53 epoxy compounds, unmodified and modified with acrylic diluent, are presented in In line with previous results, the addition of acrylic diluent produced a general increase in tensile strength with respect to both unmodified resins. Referring first to the compounds based on Epidian 53 epoxy resin, the beneficial effect in terms of strength growth was steadily reduced by increasing the diluent content. What is different is the influence of the acrylic diluent content on Epidian 5 epoxy resin: the greatest value in strength was achieved at a 5 g content of this diluent. This value was even greater than that measured on the compound based on the more resistant Epidian 53 epoxy at the same diluent content. At the greatest (10 g) and especially at the lowest (1 g) amounts of acrylic diluent, compounds based on Epidian 5 epoxy displayed lower tensile strength values.In The positive effect on tensile strength that was due to the addition of a diluent in both epoxies was confirmed also in the case of the extraction diluent. The increase in tensile strength was simultaneous with the increase in the amount of this diluent in the case of the systems based on Epidian 5 epoxy, the greatest value being obtained at a 10 g content of extraction diluent. On the other hand, the highest value of tensile strength was obtained at 5 g of diluent in compounds based on Epidian 53 epoxy resin. At the lowest (1 g) and at the greatest (10 g) amounts of extraction diluent, limited values of tensile strength were recorded, these values remaining above that measured on the pristine Epidian 53 resin. The results of the last part of the investigation can be summarized as follows: (i) the addition of the selected diluents in both epoxy resins produced general improvements in their tensile strength; (ii) the effect of the diluent content on tensile strength did not follow a precise trend; and (iii) not always did the compounds realized with the most resistant resin  develop higher strength values than those produced starting from the less resistant epoxy .From the wide and attentive investigation described so far, it was possible to conclude that the different diluents under analysis were able to affect to a certain extent the mechanical characteristics (measured in compression and tensile mode) of two commercial epoxy resins, a standard bisphenolic epoxy (Epidian 5) and a styrene-modified epoxy resin (Epidian 53), in which they were added in amounts never exceeding 10 g per 100 g of resin. The mechanical characteristics analyzed, i.e., modulus and strength, were generally enhanced upon the addition of diluents, irrespective of their chemical nature and added amounts, with the exception of compressive modulus. According to , if a noCompressive modulus, on the other hand, was differently influenced by the introduction of a diluent depending on the kind of resin and the type and content of diluent. A decline in compressive modulus was observed when epoxy, bituminous and nitro diluents were added to the resin initially characterized by a higher value of compressive modulus, i.e., Epidian 5 epoxy resin; only acrylic and extraction diluents, added at greater contents, led to an increase in the modulus measured in compression mode. The effect of the different diluents was even less clear in the case of the compounds based on the styrene-based epoxy, i.e., Epidian 53; for this resin, however, a positive effect of the different diluents on compressive modulus was generally observed at the lowest diluent content, i.e., 1 g, with the exception of epoxy diluent.As for all the measured mechanical characteristics, i.e., compressive modulus and strength and tensile modulus and strength, no precise trends can be attributed to the amount of added diluents. Referring to their chemical nature, it was noticed that the highest value of compressive strength  was obtained upon the addition of nitro diluent in the lowest amount to Epidian 5 epoxy resin . This diIt must be emphasized, however, that the optimal value of the mechanical characteristics that an epoxy resin/adhesive must possess depends on the specific application. Furthermore, alongside the characteristics analyzed, the effect that the presence of a diluent may have on the glass transition temperature of the epoxy resin and on its functional characteristics must be taken into account to identify the most suitable composition for the relevant application. In this regard, it is planned to investigate the influence of the same diluents on the thermal properties of both epoxy resins, i.e., their glass transition temperature and the residual heat of cross-linking reactions that the epoxy resins could exhibit, being cold-cured .The obtained results are substantially in line with those reported by Ozgul and Ozkul  that empFrom what was just outlined, it can be concluded that the research on this topic must be continuously updated because of the variety of resins, hardeners and new diluents that can be continuously proposed for specific applications.In the present paper, the results of a mechanical investigation performed in compression and tensile mode on two epoxy resins, cold-cured by a polyamide curing agent, upon addition of different diluents, were analyzed. The epoxy compounds were based on Epidian 5 (a bisphenolic epoxy) and Epidian 53 (an epoxy modified with styrene) resins that were modified with epoxy, bituminous, nitro, acrylic and extraction diluents added into the epoxies in different amounts, never greater than 10 g per 100 g of each resin. The cylindrical or dog-bone specimens of the resulting epoxy compounds, cold-cured at 22 °C for two weeks, were subjected to compressive and tensile tests in accordance with ISO 604 and ISO 527-1 standards, respectively. On the basis of the analysis of the results of mechanical tests, the following conclusions can be drawn.The effect of the different diluents on the compressive modulus of the epoxy resins under investigation was found to depend on the kind of resin, on the added diluent and on its content: it was not possible to identify a general trend valid for all systems. In the case of the epoxy compounds based on Epidian 5 epoxy resin, characterized by a higher value of compressive modulus in its unmodified formulation, the addition of diluents caused a decrease in this characteristic, with the exception of acrylic and extraction diluents when added in large amounts. When analyzing the epoxy compounds based on Epidian 53 resin, i.e., the styrene-based epoxy displaying a lower initial compressive modulus, increases in Ec value with respect to unmodified resin were recorded upon the addition of epoxy diluent in the largest amount , bituminous diluent up to 5 g and nitro, acrylic and extraction at the lowest amount .More clear was the resulting effect of the same diluents on the compressive strength of both epoxies: their addition produced, in fact, general improvements in this characteristic, especially for the bisphenolic Epidian 5 resin at the lowest (1 g) diluent content. Epidian 53 epoxy resin, on the other hand, displayed the highest compressive strength upon addition of 5 g of nitro diluent. Referring to modulus and strength measured in tensile tests, both properties were generally improved with the addition of diluents. The initial characteristics of the unmodified resins mostly influenced the tensile modulus of the epoxy compounds containing diluents, i.e., greater values of tensile modulus were recorded on systems based on the stiffer (Epidian 5) resin, irrespective of the type and content of the diluent; on the other hand, the tensile strength developed by the different compounds did not always reflect the strength value offered by the resin with which they were made. Referring to the quantity of diluent added to each resin, the influence of this parameter on the tensile strength was not clear; the greatest tensile modulus values were recorded with the addition of low quantities (1 g and 5 g) of diluent, even though this was not a rule respected in all cases. As a continuation of the study, the influence of the same diluents on the thermal properties of both epoxy resins will be investigated, in particular their glass transition temperature and the residual heat of the crosslinking reactions that the epoxy resins could exhibit, being cold-cured.The results of this study allow us to identify the type of diluent and its optimal quantity to obtain epoxy adhesives/components with mechanical properties suitable for a specific application.


Danio rerio) transgenic lines reporting in vivo the activity of twelve signalling pathways involved in embryonic development. Wnt/β-catenin and Jak/Stat3 were found as key pathways significantly dysregulated in both human and zebrafish samples. This information was used in a chemical-genetic analysis to test drugs targeting Wnt/β-catenin and Jak/Stat3 pathways to rescue a set of FGR phenotypes, including growth restriction and cardiovascular modifications. Treatments with the Wnt/β-catenin agonist SB216763 successfully rescued body dimensions, cardiac shape, and vessel organization in zebrafish FGR models. Our data support the Wnt/β-catenin pathway as a key FGR marker and a promising target for pharmacological intervention in the FGR condition.Foetal Growth Restriction (FGR), previously known as Intrauterine Growth Restriction (IUGR), is an obstetrical condition due to placental insufficiency, affecting yearly about 30 million newborns worldwide. In this work, we aimed to identify and pharmacologically target signalling pathways specifically involved in the FGR condition, focusing on FGR-related cardiovascular phenotypes. The transcriptional profile of human umbilical cords from FGR and control cases was compared with the response to hypoxia of zebrafish ( Therefore, it is important to start early antenatal monitoring to quickly recognize these foetuses and institute timely obstetrical interventions to reduce the perinatal deaths (Intrauterine Growth Restriction (IUGR), more recently defined as Foetal Growth Restriction (FGR) , refers l deaths .FGR is known to lead to short-and long-term consequences, in the neonatal period and adulthood, like cardiovascular, respiratory, renal, immunological, neurological and metabolic disease including, but not limited to, hypertension, diabetes, obesity and dyslipidaemia, hence representing a major burden on individuals, society and economy .At present, there is a lack of adequate intrauterine diagnostic approaches and therapeutic interventions in the paediatric period. Thus, dissecting the molecular and cellular mechanisms underlying FGR is a great challenge to improve diagnosis, prognosis and targeted treatment of this clinical entity.D. rerio), mostly by inducing hypoxic conditions in the embryo/foetus, thus faithfully mimicking placental insufficiency, without alteration of the maternal health status  as an important and targetable player in the hypoxia-induced growth retardation . More reThese studies, corroborated by growing evidence on IGF signalling involvement in human FGR , paved tin vivo, in an intact animal, under selected genetic or environmental conditions, including hypoxia or drug treatments : 4 with AGA  pregnancy, considered as healthy controls, and 4 with FGR pregnancy. Written informed consent was obtained from each woman before the enrolment, and the protocol (P1826) was approved by the Padua University Hospital Committee for Research on Human Subjects; the study was conformed to the Declaration of Helsinki. Umbilical cord samples were collected and stored in TRIzol reagent  until total RNA extraction. Sample features are reported in the Results section.D. rerio (zebrafish) were kept in a temperature-controlled (28.5°C) environment in a 12:12 light-dark cycle, staged according to Kimmel  and with permission for animal experimentation from the Ethics Committee of the University of Padova (OPBA) and the Italian Ministry of Health (Authorization number 407/2015-PR). o Kimmel  and maino Kimmel .https://zebrafish.org/documents/faq.php). Wild-type lines used in this work included Tuebingen, Giotto and Umbria strains  was added to the fish water at 0.16 mg/ml or 0.3 mg/ml, respectively. The Tricaine overdose for zebrafish euthanasia was performed as recommended by the Zebrafish International Resource Centre ia21 (ia4Tg(7xTCF-Xla.Siam:GFP) (iaTg(7xSRE-HSV.Ul23:EGFP)28 (Tg(Hsa.CTGF: EGFP)ia48 (Tg(12xSBE:EGFP)ia16 (Tg(2xID1BRE:nlsmCherry)ia17 (Tg(6xCRE:EGFP) (Tg(12xNREbglob:EGFP) (Tg(dusp6:d2EGFP)pt6 (Tg(12xGli-HSVTK:nlsmCherry)ia10 (Tg(9xGCRE-HSV.Ul23:EGFP)ia20 (Tg(5xERE:GFP) (Tg(tg:EGFP-myl7:EGFP)ia300 (Tg(fli1:EGFP)y1 (The following transgenic lines were used: Hif/Hypoxia reporter line )ia21 , Wnt/β-cGFP)ia4 , Jak/Staia28 , Hippo/Y)ia48 , TGFβ re)ia16 , BMP rep)ia17 , CREB reRE:EGFP) , Notch rob:EGFP) , FGF repP)pt6 , Sonic H)ia10 , Glucoco)ia20 , OestrogERE:GFP) , thyroidia300 , and vasFP)y1 .Zebrafish embryos were obtained from adults of the corresponding transgenic lines; transgenic fishes were outcrossed with wild type individuals to ensure fluorescent signal homogeneity in heterozygous offspring. Fluorescent embryos at 24 h post fertilization (hpf) were incubated for 2–5 days at 28°C into a hypoxic chamber  with controlled and constant 5% oxygen concentration . As controls, fluorescent sibs were kept at 28°C outside the hypoxic chamber, under normoxic conditions.igfbp1a and igfbp1b genes in hypoxic (oxygen at 2 mg/L) and normoxic conditions: 24 h of hypoxia, starting from 1 hpf; 24 h of hypoxia, starting from 24 hpf; 24 h of hypoxia, starting from 48 hpf , after rotocols . At leashttp://primer3.ut.ee), with optimal annealing temperature at 60°C. Results were expressed as relative mRNA abundance and normalized to human GAPDH or zebrafish gapdh as endogenous reference genes. The used primers are reported in Total RNA was extracted using the TRIzol Reagent , following manufacturer’s instructions. Briefly, 100 μg of umbilical cord tissue obtained from human patients and pools of 30 zebrafish embryos for each experimental condition were homogenized in 1 ml of TRIzol Reagent using 5-mm steel beads and 3 cycles of 3 min at 30 mHz in a TissueLyser instrument . 1 μg of total RNA was used for cDNA synthesis with FIREScript RT cDNA Synthesis KIT , according to the manufacturer’s protocol. The obtained cDNA was used for gene expression analysis by quantitative real-time PCR (qPCR), focusing on pathway-specific markers . qPCRs were performed in triplicate with EvaGreen method using 5 x HOT FIREPolEvaGreen qPCR Mix Plus  and Bio-Rad CFX384 qPCR System , following the manufacturer’s protocol. All primers were designed using the software Primer3 . Zebrafish larvae at 4 dpf (days post-fertilization) were placed in 48-well plates, with one larva per well in 1 ml of fish water. After 20 min of acclimation, movements of larvae were recorded repeating three cycles of 10 min of light and 10 min of dark, for a total duration of 60 min, as previously described .Zebrafish were imaged under a dissecting microscope (Leica M165FC) equipped with a CCD camera (Leica DFC7000T) and controlled by Leica Application Suite . Embryos at 3 dpf were anesthetized with 0.16 mg/ml Tricaine in fish water to facilitate the body length measurement. Heart rates were determined at 3 dpf by counting the number of atrial contractions during 60-s timeframes. The number of heartbeats per unit of time was expressed as beats per minute (bpm).Zebrafish embryos, under normoxic or 5% hypoxic conditions, were treated for 48 h with 40 µM SB216763 or 5 µM XAV939 , directly dissolved in fish water, to activate (SB216763) or inhibit (XAV939) Wnt/β-catenin signalling. Likewise, Jak/Stat3 signalling modulation was carried out by using 50 μM Tyrphostin AG490 , a knownt-test, while multiple comparisons were analysed by one-way ANOVA followed by Tukey’s test (Graph Pad Prism V7.0 software). In the charts, error bars display standard errors of the mean. The significance threshold for the p-value was 0.05. Asterisks indicate significant differences from controls. Correspondence between asterisks and significance levels is indicated in the figure legends.Signal quantification and morphological analysis were performed using the Measurements option of the Volocity 6.0 software . Pairwise analysis was carried out by unpaired To investigate if the FGR condition could induce the modification of a set of signalling pathways, considered for their known relevance in animal development and environmental response, we collected a panel of umbilical cord samples from human FGR foetuses and healthy (AGA) controls .IGFBP1 showed significant downregulation, while the IGFBP7 gene was slightly upregulated (p = 0.07) (VEGFA (p = 0.08)  , suggest = 0.07) , we dete = 0.08) .Overall, both control pathways, IGF and Hif/Hypoxia signalling, showed differential activity in human FGR samples when compared to normal (AGA) controls, similarly to what already demonstrated in hypoxia-induced zebrafish FGR models. We did not detect any significant change in the expression of pathway members for Oestrogens and Glucocorticoids (not shown) as well as for CREB , when coThe pathway screen on human samples gave us strong evidence of signalling dysregulation and/or adaptive stress response in FGR foetuses and prompted us to verify if these variations could be recapitulated in a hypoxia-induced zebrafish model for the FGR condition. Indeed, such comparison could provide some indications on which mechanisms are more robustly conserved between the two species, and globally modified when animal growth is restricted.igfbp1a were significantly upregulated in 1 and 2 dpf zebrafish embryos, with subsequent decrease at 3 dpf, while for the other paralog igfbp1b the expression was initially decreased (at 1 dpf), followed by a slight increase , also considering that this developmental time corresponds to human stages likely unaffected by placental insufficiency . We incubated zebrafish embryos in 5% hypoxia from 1 dpf and analysed gene expression at 3 dpf. When re-evaluating egulated , suggestAfter applying our protocol of long-term hypoxia, we morphologically inspected the treated embryos, observing growth restriction. Hypoxia-incubated embryos displayed, all at statistically significant levels, decrease of the body length, non-isometric reduction of the ocular region, and lack of yolk absorption , all of Tg(tg:EGFP-myl7:EGFP)ia300. By analysing its fluorescent pattern at 6 dpf, we could detect a substantial enlargement of the heart (22%) and, as deduced from the rough halving of the fluorescence after hypoxia treatment, a significant thinning of the expanded myocardial layer in the heart chambers (atrium and ventricle) (y1Tg(fli1:EGFP), where we observed a global disorganization, both in orientation and ramification, of intersomitic vessels in hypoxia-treated larvae, compared to untreated siblings  . For thesiblings . OverallTo complete the morpho-functional characterization of the zebrafish FGR models, we took into consideration their locomotor performances as well. After a 2-days incubation in 5% hypoxia, 4-dpf larvae were imaged for 1 h under alternating light/dark periods. This test allowed to detect a normal response to light stimuli in hypoxia-treated larvae, but with a significant reduction of the total distance swum, compared to untreated siblings , suggestbona fide models of FGR condition, in terms of IGF signalling dysregulation and delayed/impaired development of anatomical and locomotor features. We thus started a large-scale screen of developmental pathways, taking advantage of a wide collection of zebrafish biosensors, available at the UniPD Zebrafish Centre, able to report in vivo, though fluorescent reporters, the activation of cell signalling cascades  were maintained for 2 days under normoxia or 5% hypoxia, then euthanized and fixed at 3 dpf; finally, each pathway reporter expression was detected by fluorescent WISH and confocal-imaged. We focused our attention on heart and pharyngeal vessels, due to the heavy involvement of the cardiovascular system in FGR; as additional anatomical district, we took into consideration the forebrain region. The Hif/Hypoxia reporter invariably confirmed the upregulation of the pathway in hypoxia-treated embryos; moreover, Wnt/β-catenin and Jak/Stat3 signalling   were alsTo elucidate the potential significance of Wnt/β-catenin and Jak/Stat3 signals as therapeutic targets in FGR, we simultaneously exposed zebrafish embryos to hypoxic conditions (2-days incubation) and specific agonists/inhibitors targeting these pathways, to analyse their combined effects on FGR phenotypes. For the Jak/Stat3 signalling pathway, we exposed both normoxia- and hypoxia-treated embryos to either the Stat3 inhibitor AG490 or the Stat3 agonist LIF. Neither the Stat3 inhibition by AG490 nor the Stat3 upregulation by LIF had significant effects in rescuing body length, eye size or pericardial effusion under hypoxia . This suTg(fli1:EGFP)y1, we could observe a rescue of the intersomitic vessels organization, ramification, reciprocal distance and diameter. Conversely, the Wnt/β-catenin inhibitor XAV939 led to an exacerbation of the vascular disorganization, further corroborating the notion that Wnt/β-catenin upregulation may represent a mechanism counteracting hypoxia-induced vascular modifications  and hypoxia response , confirmed their differential expression in both species, supporting the reliability of this experimental setup.A subset of pathways, represented by CREB, Oestrogen and Glucocorticoid signalling, were undetectable or unmodified in samples from both species. This was an unexpected result as steroid hormone activities, by the way strongly interlaced with CREB signalling  and hypoThe remaining pathways, representing half of the considered collection, displayed significant changes between FGR and control cases. A set of pathways  were variably modified not only based on FGR versus control comparisons, but also depending on the species and tissue types, suggesting a complex and dynamic regulation of these signals, with organ-or organism-specific up/down-regulation. For most of these pathways the link with FGR has not been fully clarified before. Bmp and TGFβ signalling have been analysed in post-natal FGR lung as fibrosis markers ; moreoveTherefore, we have verified whether the pharmacological up- or down-regulation of Wnt/β-catenin or Jak/Stat3 signals might rescue or further exacerbate body growth indicators and cardio-vascular parameters in our zebrafish FGR models.The chemical modulation of Jak/Stat3 signalling could not significantly modify the hypoxia-induced morphological phenotypes, suggesting a limited role of this pathway on body growth and pericardial homeostasis. However, an involvement of Jak/Stat3 on other physiological parameters has not been investigated and thus could not be excluded; this is especially relevant considering that decreased STAT3 expression in placenta has been proposed to contribute to abnormal trophoblast function in idiopathic FGR-affected pregnancies . Also, Jigfbp1a/igfbp1b indicates that the rescuing effects of the Wnt/β-catenin pathway may be exerted, at least in part, by IGF signalling modulation. These findings suggest that sustaining an already hyper-activated Wnt/β-catenin signalling in FGR may represent a rational intervention to support body growth and vascular development, while counteracting cardiac alterations such as the pericardial effusion.Concerning the Wnt/β-catenin signalling, the up- or down-regulation of this pathway in zebrafish could instead significantly affect body growth, pericardial size and vascular morphology under hypoxia, leading to an ameliorated or worsened phenotype in case of agonist or inhibitor treatment, respectively. Of note, a preliminary analysis of IGF signalling members Notably, the Wnt/β-catenin agonist used in this study-(SB216763)—is a drug already applied in human therapy, especially at the endothelial level, proven to reduce infarct size and prevent cardiac ischemia . FurtherThe discovery of the involvement of the Wnt/β-catenin pathway as a signal differentially dysregulated in both human and zebrafish FGR models points to this pathway as a robust marker for Foetal Growth Restriction. Moreover, the successful rescue of FGR-related phenotypes such as body dimension, cardiac shape and vessel organization, mediated by the Wnt/β-catenin agonist SB216763, supports this pathway as a promising target for pharmacological intervention in FGR.


Alpl, Bglap, Runx2, and Sp7, and the formation of bone nodules. Triptonide is a transcription inhibitor of Wnt target gene, which diminishes C91-induced osteoblast differentiation in a dose-dependent manner. Meanwhile, C91 also induces autophagy through autophagosome formation and conversion of autophagy biomarker LC-3I into LC-3II. Autophagy inhibitor 3MA partially reduces C91-induced osteoblast differentiation and mineralization; autophagy inducer Rapamycin increases the expression of β-catenin to promote osteogenic differentiation, but cannot alleviate the inhibition of Triptonide on C91-induced osteogenic differentiation, indicating the crosstalk of canonical Wnt signaling and autophagy regulates C91-induced osteoblast differentiation. Furthermore, in order to simulate the in vivo detection of C91 in osteogenesis process, we made a C91 slow-release hydrogel with our newly established polycaprolactone and cell-integrated 3D printing system (PCCI3D module). The sustained release C91 promotes the differentiation and mineralization of ST2 cells. C91 can also enhance the proliferative activity of ST2 cells. The release rate of C91 from hydrogel gradually decreases within 7 days. During this period, the C91 is released by 83.0% and the cell viability maintained at 96.4%. Therefore, the small molecule Wnt agonist C91 promotes osteogenesis through caonical and autophagy-mediated Wnt signaling pathway with an option for translational application.The discovery and application of small molecules is one of the practical strategies of safe osteogenic drugs. The small molecule CHIR99021 (C91) is a highly specific, safe, and most effective GSK-3β Inhibitor. This study found that it efficiently activates the canonical Wnt signaling of bone marrow stromal cell ST2 and promotes osteoblast differentiation and mineralization. C91 increases the production and biochemical activity of osteoblast marker alkaline phosphatase, the expression of osteoblast marker genes  Clinically, osteogenic drugs teriparatide (PTH), abaloparatide (PTHrP) and romosozumab (sclerostin antibody) , 2 are aWnt signaling is the top of the five developmental signaling pathways, which is popular and most fruitful . Wnt sigMultiple GSK-3α/B kinase inhibitors were shown to increase bone mass. Lithium is a GSK-3β kinase inhibitor, and increases bone mineral density in a study of 26 patients treated with LiCl for more than 10 years . TreatmeIt is necessary to search for new agents of GSK-3β inhibitors for clinical use. Another FDA-approved GSK-3β inhibitor Tideglusib, commonly used in the clinical treatment of Alzheimer’s disease, resulted in a significant increase in new bone formation of fractured mice along with enhanced cortical bone bridging and medullary bone deposition. However, the drug produces an increase in serum aminotransferase levels . In addiin vitro activity. But this ATP binding site is highly conserved among more than 500 kinases, and the selectivity of ATP competitive inhibitors is poor. So, C91 is by far the most potent and specific GSK-3 inhibitor against GSK-3α、β subunits with miraculous effects (CHIR99021 (C91) is a highly selective GSK-3 inhibitor, does not inhibit CDKs . Most of effects . Howeverin vivo osteogenic function by our newly-established technology of integrated 3D printing of hard materials and cells, as well as with a drug sustained-release system.Recently, autophagy showed an involvement in osteogenic differentiation and bone formation induced by Wnt signaling , Wnt signaling inhibitor Triptonide (Tript), and autophagy inhibitor 3MA were purchased from MedChemExpress . Lyophilized gelatin methacryloyl (GelMA) and photo-cross-linking agent (LAP) from Sunp biotech , polycaprolactone (PCL) from Sigma , Trizol from Invitrogen , and Alizarin red S staining Kit from Solarbao Biotechnology .® Green Pro Taq HS Master Mixed qRT-PCR Kit II AG 11702, and reverse transcription reaction kits AG11705 from Accurate Biotechnology , and CCK-8 assay kit from MedChemExpress .BCIP/NBT alkaline phosphatase (AP) color-rendering kit, live/dead viability assay kit, alkaline phosphatase activity assay kit, DAPI staining solution, and monodansycadaverine (MDC) staining kit were obtained from Beyotime Biotechnology , AG RNAex Pro Reagent AG21101, SYBRpolyclonal anti-β-catenin antibody, GAPDH antibodies and polyclonal anti-LC-3I/II antibody were supplied by Wanlei , and FITC labeled goat anti-rabbit IgG (H+L) from Beyotime Biotechnology .Penicillin, α-MEM, and streptomycin were purchased from Gibco , fetal bovine serum (FBS) from Biological Industries (Israel), Wnt3a-expressing cells and control L cells from the American Type Culture Collection (ATCC) . ST2 cells were kindly obtained from Dr. Steve Teitelbaum, Washington University.4 cells.The cell culture of murine cell line ST2, Wnt3a-expressing cells, and control L cells was performed as previously reported . These cvia its binding to the C-terminal transactivation domain of β-catenin to stop β-catenin activity  for 72 hr until the end of the experiment.C91 was used to trigger the Wnt signal pathway of ST2 cells. In the meantime, ST2 cells treated by DMSO was used as the control. Triptonide was utilized to obstruct the Wnt signaling pathway activity . To stim5 ST2 cells were each mixed with 0.25 mL of α-MEM. Cells and C91 were individually mixed with an equal volume of GelMA solution for 3D bioprinting. The cell-loaded GelMA solution and the C91-loaded GelMA solusion were reciprocally printed out at 25°C as cell beams in a diameter of 300μm with an interval of 500μm between cell beams at a printing speed of 5 mm/s  were performed. Briefly, total RNA was extracted from the cells with Trizol as previously described . The cDNThe Alkaline Phosphatase Staining (AP staining) was performed as previously described . After 3The AP biochemical activity assay was performed as previously described . After 0ST2 cells were cultured on the 24-well plate treated with C91 or DMSO. After 48 hours, they were washed with PBS, fixed with 4% paraformaldehyde, permeabilized for 30 min with PBS containing 0.25% Triton X-100 (PBS-T), and blocked with 1% BSA in PBS. Immunostaining was performed using the rabbit polyclonal anti-mouse β-catenin antibody (1:500), and FITC labeled goat anti-rabbit IgG (H+L) secondary antibody (1:500) as described. Samples were washed three times in PBS, the last one of which included a 5 min incubation in DAPI (1:5000), and washed again  was performed to assess late mineralization . ST2 celAs previously reported . Cells wAs previously reported that MDC staining was commonly used for the detection of autophagosomes . The celAs previously reported . C91 solAs previously reported . ST2 celP <0.05 was considered a significant difference.Statistical analysis was performed by GraphPad Prism 8.0.1 software. Each experiment was repeated three times independently. The data was presented as mean ± SD. The normal distribution of dates was confirmed using Normality and Lognormality Tests. One-Way ANOVA was used to analyze the difference between multiple groups. Meanwhile, Brown-Forsythe and Welch ANOVA tests are used for correction analysis. A T-test was performed among two comparable groups. A nonparametric test was used when the above statistical methods didn’t work. Alpl, compared with the control group. C91 at 2.5 and 5 μM dose-dependently increased osteoblast differentiation, and C91 at 7.5 μM was not continuing to increase Alpl gene expression  stabilized the expression of β-catenin protein compared with the control  and Wnt target genes (Lef1 and Axin2) were all decreased at C91 and Triptonide conjunction treatment (Triptonide (Tript) is a specific blocker of the Wnt/β-catenin signaling pathway, and Triptonide blocks the Wnt/β-catenin signaling pathway through the C-terminal transactivation domain of β-catenin . ST2 cellc3b in the C91 group was higher than that in the control group  was lower than that of the C91 group , a transcription inhibitor of Wnt target gene, on C91-induced osteogenic differentiation of ST2 cells was determined. Triptonide inhibited C91-enhanced AP biochemical activity in a dose-dependent manner  system  system  to simulWe first examined the biological activity of the PCCI3D module. The PCCI3D modules were cultured for 1, 4, and 7 days, followed by Calcein AM and PI staining for live/dead cell experiments. ST2 cells in both experimental and control groups exhibited high cell viability at 96.4%  by our newly developed hard materials and cell integrated 3D printing technology, simulating osteogenesis Although C91 inhibits GSK-3 activity and activates canonical Wnt signaling, reports that C91 directly promotes osteoblast development and differentiation have not emerged. GSK-3 is a protein kinase that phosphorylates a range of substrates and plays a key role in a variety of human diseases. Examples include glycogen synthase (type II diabetes), NFkB/CBP transcription (inflammation), AP-1/NFAT (cardiac hypertrophy), and Tau/APP  as well as Wnt and Notch signaling . After clarifying the role of GSK-3 in insulin signaling, several inhibitors that are effective, specific, and do not inhibit CDKs have been developed, such as C91 , 35 and Specific inhibition of GSK-3β by small molecules is key to the screening of this class of small molecules. C91 is the most effective and specific inhibitor of GSK-3. It reduced the intensity of CDK2-cyclin A inhibition by nearly 50 times and did not inhibit any other protein kinase at 1 µM . OsteoblMDC staining using markers LC-3I/II and autophagosomes demonstrated that C91 induces autophagy during osteoblast differentiation and mineralization. In this study, we innovatively found that canonical Wnt signaling activates autophagy and is involved in the regulation of osteoblast differentiation. Autophagy can degrade damaged organelles and foreign bodies to form phosphate, which is secreted into the cytoplasm, providing components for biomineralization . AutophaAlthough C91 activates canonical Wnt signaling and is able to determine cell fate, it is not an oncogene for bone organs. Among GSK-3 inhibitors, C91 is less cytotoxic. Because the Wnt/β-catenin signaling pathway is essential for many biological responses, targeting the safety of this pathway is the primary precondition for development for therapeutic purposes . For WntC91 at concentrations of 5 μM and below had no effect on the proliferative activity of ST2 cells, with only 7.5 and 10 μM of C91 elevating cell proliferative viability , and Chongqing Science and Technology Commission—Basic Science and Frontier Technology Key Project cstc2015jcyjBX0119 (XT). The Chongqing Postgraduate Research and Innovation Project .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Previous studies have suggested that MED28, a Mediator subunit involved in transcriptional regulation, is associated with the growth of colorectal cancer cells; however, its role in the progression of metastasis such as epithelial–mesenchymal transition (EMT) and cell migration of colorectal cancer is unclear at present. The aim of this study was to investigate a potentially suppressive effect of calcitriol, 1,25-dihydroxyvitamin D3 2D3), a bioactive form of vitamin D, and the role of MED28 in the progression of EMT in human colorectal cancer cells. Suppression of MED28 increased the expression of E-cadherin and reduced the expression of several mesenchymal and migration biomarkers and Wnt/β-catenin signaling molecules, whereas overexpression of MED28 enhanced the EMT features. Calcitriol suppressed the expression of MED28, and the effect of calcitriol mirrored that of MED28 silencing. Our data indicate that calcitriol attenuated MED28-mediated cell growth and EMT in human colorectal cancer cells, underlining the significance of MED28 in the progression of colorectal cancer and supporting the potential translational application of calcitriol.Inadequate vitamin D status may increase the risk of developing multiple types of cancer. Epidemiological studies suggest an inverse association between 25-hydroxyvitamin D During tastasis .3 , a natural form of vitamin D, through a series of reactions upon sun exposure. Dietary sources of vitamin D are limited to mainly fortified foods and fish oils. After two hydroxylation events at C-25 and C-1 of cholecalciferol in the liver and kidneys, respectively, our body can produce 1,25-dihydroxyvitamin D3 2D3), i.e., calcitriol, the biologically active form of vitamin D. The genomic action of calcitriol is mediated through binding to a heterodimer, vitamin D receptor (VDR) and retinoid X receptor (RXR), two steroid receptor family members [In vitro studies have shown anticancer effects of calcitriol, including antiproliferation, prodifferentiation, and apoptosis [3 (25(OH)D3) and cancer, including colorectal cancer [Skin cells produce vitamin D members . The com members . Calcitr members . In termpoptosis , 9. Vital cancer . Therefol cancer .β-catenin signaling in human colorectal cancer cells [Mediator subunit MED28 exhibits multiple cellular roles, including facilitating transcriptional activation  and inteer cells  as well er cells , 19. Howβ-catenin signaling molecules, whereas overexpression of MED28 affected EMT in an opposite manner. Calcitriol suppressed the expression of MED28, and the effect of calcitriol mirrored that of MED28 silencing. Moreover, calcitriol could reverse the stimulatory effect of MED28 on EMT. Our data indicate that calcitriol attenuated MED28-mediated cell growth and EMT in colorectal cancer cells, reinforcing the implication of MED28 in the progression of colorectal cancer and supporting a promising, clinical application of calcitriol.The aim of this study was to investigate the role of MED28 in the progression of EMT and a potentially suppressive effect of calcitriol, the bioactive form of vitamin D, on MED28 and EMT in human colorectal cancer cells. Suppression of MED28 by RNA interference upregulated E-cadherin, an epithelial marker, but downregulated several mesenchymal biomarkers as well as Wnt/2, respectively. For RNA interference experiments, cells were transfected with MED28-specific small interfering RNA pools  using Lipofectamine™ transfection RNAiMAX Reagent. For overexpression experiments, cells were transfected with Flag-MED28 cDNA  using Lipofectamine™ 3000 Transfection Reagent.The chemicals and cell culture supplies stated in the current study were purchased from MilliporeSigma  and Invitrogen , respectively, unless stated otherwise. Human colorectal cancer cell lines, HT29 and SW480 , were cultured in 10% fetal bovine serum-containing Dulbecco's modified Eagle's medium and Leibovitz's L-15 medium without COβ-catenin, and p-GSK3β (Ser9) antibodies from MilliporeSigma ; fibronectin, E-cadherin, MMP2, and MMP9 antibodies from Abcam ; anti-Flag-tag and anti-p21 from Proteintech Group, Inc. ; antibodies for vimentin, Snail, Slug, Twist, ZEB2, proliferating cell nuclear antigen (PCNA), α-tubulin, β-actin, vinculin, and GAPDH from Santa Cruz Biotechnology ; and anti-c-Myc, anticyclin D1, and goat antirabbit or antimouse-conjugated horseradish peroxidase secondary antibodies from GeneTex, Inc. .After the addition of 100 nM of calcitriol  for 48 h or transfection for 24 h (overexpression) or 72 h (RNAi), HT29 or SW480 cells were washed with PBS briefly, and the preparation of total cell lysates was as stated before . CellulaFollowing the addition of 100 nM of calcitriol for 48 h, HT29 or SW480 cells were washed with PBS briefly, and the protocols of the RNA extraction and cDNA synthesis using Total RNA Mini Kit  and iScript™ cDNA Synthesis Kit , respectively, were as described before , 22. We 6) to their flank regions, followed by random allocation into three groups, with four mice in each group. Two weeks later, each mouse was intraperitoneally administered with the, respectively, assigned dose, 0 μg (vehicle), 0.5 μg, or 1 μg of calcitriol every other day. After 4 weeks, these mice were sacrificed, and their tissue sections and proteins were subjected to immunohistochemical or Western blotting analysis.The animal study with the approved protocol (CJCU-104-001) issued by the Institutional Animal Care and Use Committee, Chang Jung Christian University , was previously described . Brieflyμm, and deparaffinized with xylene. Endogenous peroxidase was quenched with 3% H2O2 followed by dehydration with ethanol and antigen retrieval in a microwave for 5 min. After blocking with 5% bovine serum albumin solution for 1 h, tissue sections were incubated with anti-MED28 antibodies (1 : 50) for 2 h followed by secondary antibodies for 1 h and 3,3′-diaminobenzidine tetrahydrochloride. Immunostaining was observed under Nikon (Eclipse 50i) light microscope at 400× and photographed.Xenograft tumors were fixed in 4% paraformaldehyde, cut with a thickness of 5 t test or the analysis of variance (ANOVA) test followed by Tukey's post hoc comparison to analyze the differences between groups as appropriate. The results were considered significantly different at p < 0.05.Data are presented as means ± standard deviation which was analyzed by Student's in vivo effect of calcitriol (bioactive vitamin D), and we found a dose-dependent tendency of calcitriol in reducing the tumor volumes and weights of the xenografts [β-catenin signaling and cell growth in human colorectal cancer cells [β-catenin signaling molecules, and cell growth in vivo. As shown in β-catenin signaling molecules, including β-catenin, c-Myc, and cyclin D1, in the HT29 xenografts  and CTNNB1 (β-catenin), but mutant APC [β-catenin signaling. We identified a role of MED28 in cell growth and epithelial-mesenchymal transition in these human colorectal cancer cells, where the effect of MED28 knockdown mimicked that of calcitriol, partially by upregulating E-cadherin as well as attenuating Wnt/β-catenin signaling and epithelial-mesenchymal transition , and glycogen synthase kinase 3β (GSK3β) [β-catenin in place, and casein kinase 1 and GSK3β phosphorylate β-catenin at the designated amino acids residues, which ultimately lead to ubiquitination and proteasome-mediated degradation of cytosolic β-catenin. Upon activation, the interaction of Wnt and receptors rescues β-catenin from disruption, stabilizes β-catenin, and allows the entry of β-catenin into the nucleus [β-catenin then forms a transcriptionally active complex with T cell factor (TCF) and lymphoid enhancer factor (LEF) and transactivates various downstream targets involved in cell growth such as cyclin D1 and c-Myc, as well as epithelial-mesenchymal transition (EMT) including fibronectin for mesenchymal adhesion and transcription factors Slug and Twist for transcriptional downregulation of E-cadherin. Therefore, E-cadherin and Wnt/β-catenin signaling are implicated in cancer development in the aspect of EMT, invasion, and metastasis [Cellular cadherin . The dis (GSK3β) . In the  nucleus . β-catentastasis .β-catenin complex is involved in the stabilization of cell–cell contact for epithelial cells [β-catenin translocating into the nucleus to transactivate the Wnt/β-catenin target genes [β-catenin signaling. Ultimately EMT could ensue if accompanied by the induction of the mesenchymal markers. Calcitriol upregulated the expression of E-cadherin  and in vitro (multiple CRC cell lines) approaches [β-catenin in SW480 cells using immunoprecipitation assay [β-catenin from interacting with TCF/LEF1, which suppresses the transcriptional activation of the Wnt/β-catenin target genes [β-catenin signaling in SW480 cells [β-catenin signaling molecules through suppressing the gene expression of MED28. Considering MED28 as a subunit of the Mediator complex and a link with Wnt/β-catenin signaling, it is intuitive to propose MED28 as a central player involved in the effect of calcitriol on the development of colorectal cancer.Calcitriol appears to exert its genomic and nongenomic effects to modulate the development of colorectal cancer through multiple avenues . For exa Figures . Vaughanproaches . Xin et on assay . The for80 cells , 20. It 80 cells . Therefo80 cells . Therefoβ-catenin signaling and controls transcription factors involved in downregulating E-cadherin and upregulating mesenchymal markers such as MMP9 and fibronectin. The growth-promoting and EMT-upregulating modes of MED28 could be repressed by the addition of calcitriol  Wnt/β-catenin signaling, one of the major players in the progression of CRC, regulates cell growth and epithelial-mesenchymal transition in CRC. (2) MED28 regulates Wnt/β-catenin signaling and the expression of its downstream targets. (3) Calcitriol suppressed the expression of MED28 and Wnt/β-catenin signaling molecules. Therefore, we propose that one suppressive effect of calcitriol on CRC may work through MED28-mediated Wnt/β-catenin signaling. Calcitriol exhibits promising clinical application. For example, Yu et al. reported that calcitriol confers radiosensitivity in colorectal cancer, suggesting a synergistic effect between ionizing radiation and calcitriol [In this study we identified a connection of MED28 with EMT and cell growth in human colorectal cancer cells such that MED28 regulates Wnt/ Figures . Calcitrlcitriol . Togethe