Associations of US indices with PEW were determined by logistic regression. Irrespective of gender, PEW vs. non-PEW patients had smaller RF, VI muscles, and RFCSA . US muscle sites  discriminated PEW from non-PEW patients, but the RFCSA compared to bio-impedance spectroscopy had a greater area under the curve , sensitivity (72.8% vs. 65.8%), and specificity (55.6% vs. 53.9%). AUC of the RFCSA was greatest for PEW risk in men  and women  (both p < 0.001). Gender-specific RFCSA values  indicated HD patients with smaller RFCSA were 8 times more likely to have PEW . The US approach enabled discrimination of muscle wasting in HD patients with PEW. The RFCSA was identified as the best US site with gender-specific RFCSA values to associate with PEW risk, suggesting potential diagnostic criteria for muscle wasting.This study aimed to assess muscle wasting and risk of protein energy wasting (PEW) in hemodialysis (HD) patients using an ultrasound (US) imaging method. PEW was identified using the ISRNM criteria in 351 HD patients. Quadriceps muscle thickness of  Muscle depletion is a clinical feature of protein energy wasting (PEW), a malnutrition state coexisting with inflammation and cachexia in chronic kidney disease (CKD) patients . CurrentNon-invasive methodologies assessing muscle wasting allow for timely treatment, but existing methods have disadvantages in terms of accuracy, cost, and feasibility ,5. The mThe present study therefore evaluated the US method to identify HD patients at risk of PEW by targeting measures of QMT and CSA. The quadriceps muscle, due to its large size and assessment ease , was theThis cross-sectional study was undertaken with maintenance HD patients screened for the Palm Tocotrienol in Chronic Hemodialysis (PATCH) study in Malaysia. Patient recruitment was conducted between September 2015 and September 2016 at four public hospitals and six non-governmental dialysis centers in the Klang Valley where patients access 4-hourly dialysis sessions three times weekly as per the standard protocol. Eligibility criteria were patients dialyzing for ≥ 3 months, aged ≥ 18 years and who had provided written consent. Patients with repeated history of hospitalizations, inter-current illnesses over six months prior to the recruitment, or diagnosed with inflammatory diseases or malignancy were excluded. The stock flow of patients included in the final analysis is presented in The study was approved by the Medical Research and Ethics Committee, Ministry of Health, Malaysia (NMRR-15-865-25260) and the Medical Research Ethics Committee of the National University of Malaysia (NN-039-2015).rectus femoris (RF) and vastus intermedius (VI) muscles were determined (CSA) was measured at the mid-thigh landmark . Standardized anatomical landmarking was performed as per the International Society for the Advancement of Kinanthropometry (ISAK) protocol  by an IStermined a on bothlandmark b. Scans Firstly, the scanning depth of the US transducer was adjusted to view the femur at the designated points, the mid-thigh and 2/3-length, and positioned perpendicular to the long axis of the thigh. The transducer was then moved along the axis of the thigh to identify the thickest width of the RF muscle with the VI and femur visualised in a single field. The identified site was centered on the femur to reflect this view and this image was captured. The QMT was drawn as “inner-to-inner” septa of the thickest region of the RF muscle, whereas the highest point from the femur to the outer septa of the RF represented muscle thickness for the VI.CSA measurement. In cases of patients with larger muscles, the panoramic view option was used to maneuver the transducer smoothly from left to right along the axis of the identified site to capture the image of the muscle septa. The inner echogenic line of the RF muscle was outlined by a moving cursor before freezing the image to calculate the RFCSA in cm2.The same protocol for QMT thickness was applied for the RF®, Apex Tool Group, LLC, NC, USA). MAMC and mid-arm muscle area (MAMA) were calculated using the Heymsfield equations [Anthropometric data were collected by the same dietitian (S.S.) to eliminate inter-observer variation. These included post-dialysis weight , body mass index (BMI), triceps skinfold (TSF) thickness as per the ISAK protocol on the dominant or non-fistula arm with a Harpenden skinfold caliper  , and midquations . The midquations .Body composition was assessed using a portable whole-body BIS device  with the patient resting in the supine position before dialysis. The hydration status, lean tissue mass (LTM), and fat tissue mass (FTM) data generated were based on the physiologic tissue model . Both leRoutine biochemistry parameters for serum urea, creatinine, and total iron-binding capacity were based on in-house laboratory analyses as per the standard operating procedures accredited by the Ministry of Health, Malaysia. Additional tests, such as serum albumin (bromocresol green method), high-sensitivity C-reactive protein (hsCRP) (particle-enhanced immunoturbidimetric assay), and bicarbonate (enzymatic method) were analyzed by an accredited external laboratory. Interleukin-6 (IL-6) measurement using an enzyme-linked immunosorbent assay was performed at an institutional laboratory (S.S.N. and B.-H.K.). All biochemistry analyses were based on mid-week-collected fasting blood samples.TM 2.2.16 .24-hour dietary records were collected for three days, inclusive of two random weekdays and one optional weekend day . Energy The malnutrition–inflammation score (MIS) rating assessed the severity of effect of the malnutrition–inflammation complex syndrome on the nutritional status . The cum®, BK-7498; Fred Sammons, Inc., Burr Ridge, IL, USA) on the dominant or non-fistula hand [Handgrip strength (HGS) was assessed using a handgrip dynamometer  < 25 kcal/kg ideal body weight (IBW).PEW was diagnosed according to these ISRNM diagnostic criteria : BMI < 2t-test and the Mann–Whitney’s U-test for normally and non-normally distributed data, respectively. Categorical data were analyzed using the Chi-squared test. The receiver operating characteristic (ROC) curve analysis was applied to US measures and physical parameters whereby the area under the curve (AUC) indicated the probability of measures to identify patients at risk of PEW. Cut-off points to discriminate the risk of PEW depended on the highest sensitivity (1-specificity) value from the ROC curve. The logistic regression analysis determined associations of these values with PEW risk and the odds ratio was adjusted for confounding variables . All the analyses were computed using the IBM Statistical Package for Social Sciences version 26.0 . Statistical significance was set at p < 0.05 for all the tested parameters.Variables were presented as mean ± standard deviation (SD), median (interquartile range (IQR)), or frequency (percentage). The normal distribution of continuous variables was assessed using the Kolmogorov–Smirnov test. Group differences were analyzed using the Student’s n = 81/351). Age, ethnicity, dialysis vintage, and dialysis adequacy did not significantly differ between PEW and non-PEW patients except for gender (p = 0.008) (p < 0.001), creatinine (p = 0.001), and albumin (p < 0.001), but higher serum bicarbonate (p = 0.009). IL-6 and hsCRP were not significantly different between groups. PEW compared to non-PEW patients had lower DEI  and dietary protein intake . MIS values were higher in PEW patients  compared to non-PEW patients.Prevalence of PEW was 23.1% (= 0.008) . PEW comp < 0.001) for PEW compared to non-PEW patients irrespective of gender  and BCM . Irrespective of gender, FTM was significantly lower in PEW patients . After the LTM and FTM were corrected for height, significantly lower LTI  and FTI  were observed in the PEW patients overall; and men with PEW . The FTI in women was affected by PEW , but not the LTI. Only the HGS of men was significantly affected by the presence of PEW .Body composition measures reflective of the muscle mass, LTM and BCM, did not significantly differ between groups. However, men with PEW compared to men without PEW had lower LTM .All US measures were significantly lower in PEW compared to non-PEW patients irrespective of gender (p < 0.001). Other measures failed to discriminate PEW from non-PEW patients  except for BIS−LTI (p = 0.029). However, the AUC sensitivity and specificity for BIS−LTI were lower compared to the RFCSA.Comparison of US measures with BIS-derived muscle measures and the HGS for risk of PEW was determined using the ROC analysis with derived AUC data presented in We next compared the AUC computed for each US measure against the PEW risk for both men and women .CSA measurement elicited the greatest AUC specific to gender  compared to other muscle sites  and < 4.47 cm2 for women   compared to those above these values (p < 0.001) even after adjustment for confounding factors (Only the RFle sites . Based oy = 80%) . Associae values . The ass factors .n = 81) when applying the ISRNM PEW diagnostic criteria [PEW, common in maintenance HD patients, greatly contributes towards morbidity and increases mortality risk. We observed for this middle-aged HD population that the prevalence of PEW was 23.1%  observed significantly smaller QMT, namely of the RF and VI muscles, in malnourished HD patients (MIS ≥ 6), as well as a significant negative correlation between MIS and US muscle sites . HoweverContrarily, applying BIS and HGS could only differentiate muscle wasting in men with PEW. This may be because men compared to women are more vulnerable to muscle wasting ,20. ThisCSA gender-specific values to facilitate screening and identification of HD patients at risk of PEW. We found the PEW risk to be 8 times higher in the HD patients with the RFCSA lower than gender-specific values for men  and women . For the purpose of sarcopenia diagnosis in the elderly (73.7 ± 9.2 years) pre-dialysis CKD population, Souza et al. (2018) also identified the RFCSA gender-specific values  using the US approach for muscle wasting [Asian Working Group for Sarcopenia has noted lower limits for low muscle mass in Asians compared to the Western population due to gender, body size, and ethnic differences [Importantly, our study allowed for the generation of clinically meaningful RF wasting . Of note wasting . Dialysi wasting . As regaCSA was the most superior site amongst the examined US measures in discriminating PEW risk compared to other physical parameters for body composition. Other studies have reported significant correlations between the RFCSA and the CT-derived LBM for upper (r = 0.286) and lower limbs (r = 0.271), BIA-derived whole-body fat-free mass (r = 0.430), HGS (r = 0.300), and physical performance measures in pre-dialysis CKD patients [CSA only constitutes 10% of the total quadriceps muscle CSA [CSA observed in our study suggests that this US site has great utility as a surrogate measure for whole-body muscle mass and physical strength.The RFpatients ,26 and cbs r = 0.1, BIA-debs r = 0.1, BIA-deer r = 0.6 and lowscle CSA . Thus, aCSA and deterioration of the quadriceps would therefore affect physical function which is also observed in PEW patients [Besides being a large skeletal muscle group in the body enabling easy assessment , the strpatients . Severalpatients ,11,30, ppatients , criticapatients ,31, and patients . Losses patients ,32 or a patients , indicatCSA gender-specific values for identification of patients at risk of PEW. However, application of these values for PEW risk to HD populations in other countries warrants further investigation. US scans in this study were completed within 15 min without hindrance to the patient’s dialysis schedule, thereby emphasizing convenience, feasibility, and non-invasive nature of the technique for assessment of muscle wasting and PEW risk.The immediate strength of the current study is that our observations are based on the largest multicenter sampling performed for US assessment of muscle wasting involving HD patients from both public and non-governmental organization dialysis centers. This large dataset enabled generation of the RFAs for the practical issues related to US imaging, the measurement is operator-dependent , but thiCSA values in predicting hospitalization or mortality risk for PEW patients could not be investigated. With four obese patients in our study, we faced difficulty in locating the femur and poor visualization of RF muscle borders. Therefore, generalization of these results to obese patients will be restricted. Further, exclusion of patients with amputation and reduced mobility may have led to selection bias, as these patients experience greater muscle wasting due to their physical inability. Information on the physical activity level, a factor that may influence muscle status, was unavailable, as it was not part of the data collected for the screening study.Limitations inherent to cross-sectional studies are also acknowledged in this study, as cause–effect relationship or prognostic ability of the RFCSA, a single site measure of the lower limbs, was a better indicator of PEW risk compared to other indirect approaches. The RFCSA was identified as the best US site and the developed gender-specific RFCSA values were associated with PEW risk. Therefore, application of a US method in a clinical setting should be considered for a rapid screening of PEW risk. Future studies investigating prospective evaluation of the US values for hospitalization and mortality risk in PEW patients as well as evaluating changes in muscle status using a US method as a targeted outcome of nutritional intervention in treating PEW is highly encouraged.Our study indicated that the US method discriminated muscle wasting in HD patients with PEW irrespective of gender. The RF


Clostridioides difficile toxin A (TcdA) has been shown to inhibit cellular Wnt signaling, the major driving force behind the proliferation of epithelial cells in colonic crypts, likely through the inhibition of β-catenin nuclear translocation. Herein, we aimed to advance the understanding of this mechanism by replicating the findings in vivo and by investigating the specific role of Rac1, a member of the Rho GTPase family, on the inhibition of the Wnt-induced β-catenin nuclear translocation triggered by TcdA. To investigate the effects of TcdA on the Wnt/β-catenin pathway in vivo, we injected the ileal loops of C57BL/6 mice with TcdA [phosphate-buffered saline (PBS) as the control] to induce C. difficile disease-like ileitis. After 4 h post-injection, we obtained ileum tissue samples to assess Wnt signaling activation and cell proliferation through Western blotting, immunohistochemistry, and qPCR. To assess the role of Rac1 on Wnt signaling inhibition by TcdA, we transfected rat intestinal epithelial cells (IEC-6) with either a constitutively active Rac1 plasmid (pcDNA3-EGFP-Rac1-Q61L) or an empty vector, which served as the control. We incubated these cells with Wnt3a-conditioned medium (Wnt3a-CM) to induce Wnt/β-catenin pathway activation, and then challenged the cells with TcdA. We assessed Wnt signaling activation in vitro with TOP/FOPflash luciferase assays, determined nuclear β-catenin translocation by immunofluorescence, measured cyclin D1 protein expression by Western blotting, and quantified cell proliferation by Ki67 immunostaining. In vivo, TcdA decreased β-catenin, cyclin D1, and cMYC expression and inhibited the translocation of β-catenin into the nucleus in the ileum epithelial cells. In addition, TcdA suppressed cell proliferation and increased Wnt3a expression, but did not alter Rac1 gene expression in the ileum tissue. In vitro, constitutively active Rac1 prevented Wnt signaling inhibition by enabling the β-catenin nuclear translocation that had been blocked by TcdA. Our results show that TcdA inhibits Wnt/β-catenin pathway in vivo and demonstrate that this inhibition is likely caused by a Rac1-mediated mechanism. Clostridioides difficile (C. difficile), a gram positive, spore forming anaerobic bacterium, is a major cause of nosocomial diarrhea (C. difficile infection are C. difficile toxin A (TcdA) and C. difficile toxin B (TcdB). TcdA and TcdB enter cells through receptor-mediated endocytosis and inhibit the small Rho guanosine triphosphatases (GTPases), such as Rho, Rac1, and Cdc42  receptor in the colonic epithelium . HoweverAll experimental protocols were approved by the Federal University of Ceará Committee on the Ethical Treatment of Research Animals (CEUA. N. 2727150218) and were performed in accordance with the Guide for Care and Use of Laboratory Animals . Male C57BL/6 mice (8 weeks old) were obtained from the vivarium of the Department of Physiology and Pharmacology of the Federal University of Ceará. The mice were maintained in a temperature-controlled environment (22 ± 1°C) with a 12-h light/dark cycle and had a free access to drinking water and standard diet.n = 6 in each group) were fasted for 4 h and had a free access to water before being anesthetized . After midline laparotomy, a 4-cm ileal loop was ligated, and 10 μg of TcdA (TechLab) in 200 μl of phosphate-buffered saline (PBS) was injected at the site of the ligations. Control loops were injected with only 200 μl of PBS. After 4 h, the mice were euthanized , and the ileal loops were removed for subsequent analysis of the parameters defined in this study.TcdA-induced murine ileitis was induced as previously described  with som−ΔΔCT) method  analysis of the gene expression of β-catenin, cyclin D1, cMYC, Wnt3a, and Rac1 was performed in mouse ileum tissue stored in RNAlater solution (Qiagen), an RNA stabilizer, at −80°C. Total RNA was extracted by an RNA isolation system (Promega) according to the manufacturer’s protocol. The RNA was quantified by a NanoDrop spectrometer (Thermo Fisher Scientific), and RNA quality was determined by examining the 260/280 ratio > 1.8. A total of 1 μg RNA was then reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer’s protocol. qPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems), as described in the manufacturer’s instructions. The sequences of the primers are listed in ) method .To obtain the nuclear extract from the ileum tissue samples, a nuclear extract kit (Abcam) was used according to the manufacturer’s protocol. Protein concentrations were determined through bicinchoninic acid assay according to the protocol of the kit manufacturer (Thermo Fisher Scientific). Fifty micrograms of reduced protein in Laemmli sample buffer with β-mercaptoethanol (Bio-Rad) was run on an SDS-PAGE gel (10%) and transferred onto PVDF membrane for 2 h. Then, the membranes were blocked with 5% blotting-grade blocker (Bio-Rad) for 1 h at room temperature and incubated overnight with anti-β-catenin  and anti-B23  at 4°C. Then, the membranes were incubated with secondary antibody conjugated with horseradish peroxidase  for 2 h at room temperature. The fluorescence of a chemiluminescent signal system was detected by a ChemiDoc™ XRS+ system , and the bands were quantified by densitometry using Image Lab 5.0 software (Bio-Rad).Sections (4 μm thick) were prepared from paraffin-embedded ileum tissue. Then, the samples were deparaffinized, dehydrated, immersed in retrieval solution  for 30 min in PT Link (DAKO), incubated with 3% (v/v) hydrogen peroxide to block endogenous peroxidase for 20 min at room temperature, and washed in PBS. The sections were then incubated with β-catenin , cyclin D1 (DAKO Flex), cMYC , Ki67 , and Wnt3a  antibodies diluted in diluent antibody solution (DAKO) for 1 h at room temperature. Next, the sections were washed with wash buffer and incubated with polymer (HRP DAKO) for 30 min. The slides were then washed and stained with chromogen 3,3′-diaminobenzidine peroxide (DAB), followed by counterstaining with Mayer’s hematoxylin, dehydrated in a graded alcohol series, cleared with xylene, and placed on a coverslip. The negative controls were processed simultaneously as described above, with the primary antibody being replaced by the diluent antibody. The images were captured by means of a light microscope coupled to a camera with an LAZ 3,5 acquisition system . One hundred epithelial cells were counted from each field to quantify the percentage of cells showing positive immunostaining for each protein.2 conditions for 21–30 passages. For all the experiments, the IEC-6 cells were released using 0.25% trypsin-EDTA for 5 min.The rat intestinal epithelial cell (IEC-6) line was obtained from the repository of cells in Rio de Janeiro. IEC-6 cells were cultured in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum (FBS), 1% antibiotics  and 0.1 U/ml bovine insulin (Sigma) at 37°C in a humidified incubator under 5% COThe IEC-6 cells were transfected with pcDNA3-EGFP-Rac1-Q61L [Addgene, the vector pcDNA3 contains the mutant Rac1 encoding cDNA (Rac1) in its active form and the green fluorescent protein (GFP); Wnt3a-CM was obtained from L cells stably transfected with the Wnt3a plasmid cultured in DMEM supplemented with 10% FBS for 4 days. The medium was collected and filtered using a 0.22 μm filter, and fresh medium was added to the cells and cultured for 3 more days. After this period, the medium was collected and mixed with the previous medium at a 1:1 ratio. As a control, the CM was similarly generated with an untransfected parental cell line.Renilla gene under the control of the constitutively active herpes simplex virus thymidine kinase promoter and was used as a normalizer  with a microplate luminometer. This assay is as a gold standard to evaluate β-catenin translocation into the nucleus.The IEC-6 cells were cotransfected with TOPflash (Millipore)/FOPflash (Millipore) and the pRL-TK vector (Promega) in combination with the pcDNA3-EGFP-Rac1-Q61 or an empty vector with Lipofectamine 3000 reagent (Thermo Scientific) according to the reagent manufacturer’s instructions. TOPflash is a TCF reporter plasmid containing two sets of three copies of wild-type TCF-binding sites and is driven by a minimal promoter of thymidine kinase inserted upstream of the luciferase reporter gene. FOPflash contains mutated TCF-binding sites driven by the same thymidine kinase promoter and upstream luciferase open reading frame as used in TOPflash. FOPflash is used as a negative control for TOPflash activity. The pRL-TK vector contains the Immunofluorescence assays were performed as previously described . Briefly5 cells/well in a 24-well plate were fixed with 4% paraformaldehyde solution for 30 min, washed with PBS, and permeabilized with 0.1% Triton X-100 for 5 min. The samples were then blocked for 1 h with 5% BSA and incubated with anti-Ki67 antibody  for 1 h at room temperature. The samples were then incubated with the HRP polymer (DAKO) for 30 min at room temperature. Then, the cells were stained with DAB chromogen (DAKO) and counterstained with hematoxylin. The coverslips were placed on slides using Faramount (DAKO). The percentage of cells showing positive immunostaining for Ki67 was determined by counting 100 hematoxylin-stained nuclei.IEC-6 cell proliferation was evaluated by Ki67 immunocytochemistry, as previously described by Lysate samples from the treated IEC-6 cells were harvested in RIPA buffer (Thermo Scientific) by the procedure described by the manufacturer. Protein concentrations were determined through bicinchoninic acid assay according to the protocol of the kit manufacturer (Thermo Fisher Scientific). Thirty micrograms of reduced protein in Laemmli sample buffer with β-mercaptoethanol (Bio-Rad) was run on an SDS-PAGE gel (10%) and transferred onto PVDF membrane for 2 h. Then, the membranes were blocked with 5% blotting-grade blocker (Bio-Rad) for 1 h at room temperature and incubated overnight with anti-cyclin D1 , anti-Rac1 , and anti-β-actin  at 4°C. Then, the membranes were incubated with secondary antibody conjugated with HRP  for 2 h at room temperature. The fluorescence of a chemiluminescent signal system was detected by a ChemiDoc™ XRS+ system , and the bands were quantified by densitometry using Image Lab 5.0 software (Bio-Rad).t-tests were used to compare two groups, and analyses of variance (ANOVAs) followed by Bonferroni’s multiple comparison test were used for comparisons of more than two groups. A value of p < 0.05 was considered significant.All quantitative results are expressed as the means ± standard error of the mean (SEM). Statistical analysis of the data was performed using GraphPad Prism software, version 6.0. Student’s in vivo. We found that TcdA downregulated (p < 0.02) β-catenin gene expression in ileum tissue compared to the level in the control  and cyclin D1 (p < 0.04) gene expression in the ilea of the treated mice compared to that in the ilea of the control group mice (p < 0.0001) cyclin D1 immunostaining in the ilea of the treated mice compared to that in the ilea of the control group mice. In the mouse ileum, cyclin D1 is mainly expressed by epithelial cells from ileal crypts (p < 0.0001) the number of cells showing cMYC immunostaining in the ilea of the treated mice compared to the number in the ilea of the control group mice  cells challenged with TcdA, which we evaluated by immunostaining Ki67, which is a marker of cell proliferation. As shown in in vivo by decreasing the translocation of β-catenin to the nucleus, and thus downregulating its target genes (c-MYC and cyclin D1). Through this pathway, TcdA was shown to inhibit intestinal crypt cell proliferation in a mouse model of C. difficile-induced disease-like ileitis despite Wnt3a upregulation observed in the crypts and lamina propria cells, which were insufficient to induce Wnt/β-catenin activation.Herein, we demonstrate that TcdA inhibits Wnt signaling in vitro even when the cells are in the presence of the activator Wnt3a, an endogenous agonist; lithium chloride, a GSK3β inhibitor; and/or z-VAD-fmk, a nonspecific caspase inhibitor (Renilla ratio), β-catenin nuclear translocation, cyclin D1 protein expression, and cell proliferation, implying that TcdA inhibits the Wnt/β-catenin pathway in a Rac1-dependent manner.Our laboratory had previously demonstrated that TcdA inhibits the Wnt/β-catenin pathway in IECs nhibitor . Here, wAs shown previously, the inhibitory effects of TcdA on the Wnt/β-catenin pathway persist even in the presence of constitutively active β-catenin , suggestAlthough Rac1 mRNA expression is not modified by TcdA, as shown in Drosophila embryos, the deletion of RacGAP50C, a gene that negatively regulates Rac1, promoted the activation of Wnt/β-catenin .CM designed and performed all the experiments, analyzed the data, wrote the manuscript, and helped in the acquisition of the data. GS, GF, and DP performed experiments and contributed to the data analysis. DC, RL, BL, and JA helped with the experimental design and the revision of the manuscript. GB, the principal investigator for the grant and for constructing the experimental design, supervised the project and helped write the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Focal adhesion kinase (FAK) and Wnt signaling pathways are important contributors to tumorigenesis in several cancers. While most results come from studies investigating these pathways individually, there is increasing evidence of a functional crosstalk between both signaling pathways during development and tumor progression. A number of FAK–Wnt interactions are described, suggesting an intricate, context-specific, and cell type-dependent relationship. During development for instance, FAK acts mainly upstream of Wnt signaling; and although in intestinal homeostasis and mucosal regeneration Wnt seems to function upstream of FAK signaling, FAK activates the Wnt/β-catenin signaling pathway during APC-driven intestinal tumorigenesis. In breast, lung, and pancreatic cancers, FAK is reported to modulate the Wnt signaling pathway, while in prostate cancer, FAK is downstream of Wnt. In malignant mesothelioma, FAK and Wnt show an antagonistic relationship: Inhibiting FAK signaling activates the Wnt pathway and vice versa. As the identification of effective Wnt inhibitors to translate in the clinical setting remains an outstanding challenge, further understanding of the functional interaction between Wnt and FAK could reveal new therapeutic opportunities and approaches greatly needed in clinical oncology. In this review, we summarize some of the most relevant interactions between FAK and Wnt in different cancers, address the current landscape of Wnt- and FAK-targeted therapies in different clinical trials, and discuss the rationale for targeting the FAK–Wnt crosstalk, along with the possible translational implications. Focal adhesion kinase (FAK) is a key regulator of growth factor receptor and integrin-mediated signaling that coordinates multiple fundamental processes such as migration, invasion, angiogenesis, cell survival, or epithelial–mesenchymal transition (EMT), in both normal and cancer cells. Due to this prominent role in cancer, a vast number of preclinical and clinical studies have explored different strategies to block FAK signaling for therapeutic purposes. Molecular and cellular mechanisms of FAK action on both tumor and stromal cell biology, regulation of its activity, and FAK-targeted anticancer therapeutic strategies that are, nowadays, under preclinical and clinical development have been extensively reviewed in different excellent review articles ,2,3,4. HWnt signaling it is one of the most fundamental and conserved regulatory systems in animals. This pathway controls many critical aspects of development and adult tissue homeostasis by regulating cell proliferation, differentiation, migration, genetic stability, and apoptosis, as well as by maintaining adult stem cells in a pluripotent state. Dysregulation of this pathway is associated with a broad spectrum of human diseases, ranging from neurodegenerative disorders to fibrosis, osteoporosis, and cancer. Consequently, Wnt-related research has become a major area for targeted drug discovery and therapeutic development ,6. For aA crosstalk between pathways occurs when the combinatorial signal from both pathways produce a different response than the one triggered by each individual pathway . It mustPTK2, is located on chromosome 8q24.3 [FAK is a non-receptor protein tyrosine kinase that mediates growth factor- and adhesion-dependent signaling through several downstream pathways leading to cell migration, invasion, cell cycle progression, and survival ,12,13, de 8q24.3  and is he 8q24.3 ,16,17,18e 8q24.3 , as welle 8q24.3 ,21. Actie 8q24.3 . For inse 8q24.3 .Although FAK itself has not been demonstrated to be an oncogene, FAK overexpression and activation have been reported in tumors of broad tissue origin , especiaFAK signaling is activated by several different mechanisms that include interaction with integrins, growth factor receptors, G protein–coupled receptors, or cytokine receptors ,33. The The Wnt signaling pathway is an ancient and highly conserved pathway that regulates crucial aspects of cell fate determination, cell migration, cell polarity, neural patterning, and organogenesis during embryonic development . It has 2+ pathways. In the Wnt/Ca2+ pathway, Wnt-Fzd binding activates phospholipase C (PLC) via G proteins, leading to diacylglycerol (DAG) production and an increase of intracellular Ca2+ levels, subsequently activating downstream effectors that include protein kinase C (PKC) [2+ levels also activate calcineurin and calmodulin-dependent protein kinase II (CaMKII) [The Wnt family consist of 19 secreted glycoproteins that transduce signals by binding to frizzled (Fzd) receptor complexes . Based o C (PKC) . The inc(CaMKII) . The PCP(CaMKII) . This pa(CaMKII) ,48. In a(CaMKII) .FAK is crucial during development, tissue regeneration and wound healing. Deletion of FAK during mouse development results in lethality at embryonic day 8.5 and a block in cell proliferation. FAK activity is also essential for developmental processes controlling blood vessel formation, cell motility and polarity . FAK-defBeyond development, Wnt signaling exerts crucial roles in adult tissues and organs, in daily processes of tissue homeostasis and regeneration, in organ repair after injury, and in adult neurogenesis . The besDrosophila ovarian morphogenesis [Xenopus laevis [There are many described interactions between FAK and Wnt during embryonic development (reviewed in ). FAK anogenesis  and in rs laevis , where Fs laevis . In addis laevis , while as laevis . FAK ands laevis . More res laevis . FAK alss laevis .Given Wnt’s essential role in embryonic development, tissue homoeostasis, and stem cell biology, this pathway must be tightly regulated; its dysregulation has been associated with many types of cancer. “No man is an island”, and similarly no pathway is modulated without affecting others . UnderstAPC) occur in 85% of the cases, while activating mutations of the proto-oncogene β-catenin (CTNNB1) occur only in 5% of the cases [Colorectal cancer (CRC) is the second leading cause of cancer morbidity and mortality worldwide . Genetiche cases . Activathe cases . While Whe cases , activathe cases .In the absence of Wnt, the canonical function of APC is to form a “destruction complex” with Axin/Axin2, CK1-α, and GSK-3β promoting the sequestration, ubiquitination, and subsequent proteasomal degradation of β-catenin . APC is min/+ mice and in human CRC tissues, in addition they promoted Wnt/β-catenin pathway activation by phosphorylating GSK3β leading to β-catenin accumulation, which in turn initiates and supports intestinal tumorigenesis. Pharmacological inhibition of FAK/PYK2 repressed adenoma formation in APCmin/+ mice and reduced intestinal levels of phospho-GSK3β and β-catenin, indicating that the FAK/PYK2/GSK3β axis is critical in APC-driven intestinal tumorigenesis [V600E-mutant CRC cell lines through FAK activation-mediated phosphorylation of GSK3. Co-targeting of BRAF/Wnt pathways or BRAF/FAK pathways exerted strong synergistic antitumor effects in cell culture and mouse xenograft models [BRAF mutations [As mentioned above, GSK3β is a central player in the canonical pathway; it operates by regulating the phosphorylation and degradation of β-catenin, and its activity is tightly controlled . Gao et igenesis . Altogett models . Given tutations .LRP5 and FZD2, and upregulated SFRP5, one of several members of the SFRP family that controls Wnt pathway signaling, specifically by preventing it from binding to its receptor [Interestingly, FAK inhibition with the small molecule inhibitor Y15 increased DKK1, a known inhibitor of the Wnt pathway that plays an important role in CSC regulation in the metastatic CRC cell line, SW620. Y15 also downregulated Wnt pathway genes, such as receptor .RNA [Another link between Wnt and FAK signaling is the finding that the expression of HEF1 (human enhancer of filamentation 1), implicated in progression of CRC, was also shown to modulate both pathways. HEF1 localizes to focal adhesions to coordinate FAK and Src signaling cascades in integrin-dependent adhesion, migration, invasion, and survival ,79. In aRNA .In conclusion, there is an unequivocal evidence that FAK and Wnt pathways play a role in regulating CRC initiation and progression. These findings suggest that pharmacological inhibition of FAK might be effective in the treatment of CRC ,82.An interesting correlation between FAK and Wnt signaling was found in malignant mesothelioma (MM), an aggressive neoplasm that develops from the mesothelial cells lining the pleural, peritoneal, and pericardial cavities . TreatmeMorus alba, the c-Src/FAK and ERK pathways were inactivated through the reduction of ROS levels. This in turn activated GSK3β, leading to the inactivation of the β-catenin transcription factor. As a result, migration and invasion of A549 cells were attenuated, demonstrating that the anti-metastatic effect of Maclurin was exerted by antioxidative activity and inhibition of Src/FAK–ERK–β-catenin signaling pathway [Momordica charantia [A relation between FAK and Wnt signaling was also found in a study evaluating the function and mechanism of FAK in regulating the inflammatory response in the A549 cell line, a model for non-small cell lung cancer (NSCLC). The inhibition of FAK decreased the activation of the Wnt and NF-κB signaling pathways, accompanied by a reduction in inflammatory activity . In anot pathway . Similarharantia .Both Wnt/β-catenin and FAK pathways were independently found to be implicated in idiopathic pulmonary fibrosis (IPF). Blocking Wnt signaling strongly attenuated and reversed fibrosis in a murine IPF model system . SimilarAPC tumor suppressor gene also plays a significant role in breast cancer carcinogenesis. APC is silenced by hypermethylation or mutated in about 70% of human breast cancers [In a model of high-grade serous ovarian cancer (HGSOC) it was recently shown that FAK activity sustained intrinsic and acquired cisplatin resistance via a Wnt/β-catenin-Myc ‘stemness’ pathway . Previou cancers . Recentl cancers . In addi cancers . Similar cancers .While in some cancer types the relationship between FAK and Wnt is already well evident, in other cancers this crosstalk is still elusive. Different studies of hepatocellular carcinoma, melanoma and acute myeloid leukemia suggest that FAK operates upstream of Wnt, while in prostate cancer Wnt seems to acts upstream of FAK. In addition, a study of renal carcinoma reveals that Wnt and FAK might act concomitantly in promoting cancer progression.WNT11 and CCDN1), accelerating the vasculogenic mimicry capacity in these cells, which is associated with a high tumor grade, invasion, metastasis, and a short survival [GOLPH3) was able to activate simultaneously the FAK/Raf/MEK pathway and Wnt/β-catenin promoting RCC cell proliferation and malignancy. However, in this scenario, whether FAK and Wnt act independently but concomitantly or interdependently from each other was not addressed [Deletion of FAK in hepatocytes blocks tumor proliferation and prolongs the survival in a c-Met/β-catenin-driven hepatocellular carcinoma (HCC) mouse model , suggestsurvival . A studysurvival . In a stddressed . A more ddressed . Anotherddressed .Despite the increasing number of new targeted anticancer agents available in the clinic, treatment resistance and failure remain major challenges in the management of most advanced solid cancers, including breast  and coloIn addition to the roles of Wnt signaling in development, dysregulated Wnt also contributes to cell proliferation, chemoresistance and enhanced tumorigenic potential of CSC, all of which are factors involved in tumor recurrence after therapy, resistance to further anticancer therapies, and poor survival . TherefoIn general, agents targeting Wnt signaling include compounds at the ligand/receptor level , transcriptional level  and at the level of Wnt secretion    binds to Sam68, decreasing the expression of β-catenin target genes. In a phase I study, CWP291 was considered safe and demonstrated single-agent activity in acute myeloid leukemia patients, with plans for future combination studies . RegardiOne of the emerging and most promising ways for targeting Wnt signaling is to block Wnt ligand production through the inhibition of the acyl-transferase Porcupine . Wnts neSeveral other small molecules antagonizing the interaction between β-catenin and TCF/LEF1, such as PNU74654, 2,4 diamino-quinazoline, PKF115-584, or CGP 049090, have been identified. Other small molecules include those targeting Dvl , CK1, AXIN, or Tankyrase 1 and 2 (that promote AXIN stabilization). Natural compounds, such as vitamin D, curcumin, quercetin, or resveratrol, or existing drugs in the market such as Sulindac and Celecoxib are also being investigated in preclinical studies for Wnt signaling inhibition .Taken together, the data and rationale behind using Wnt inhibitors supports them as promising therapeutics. Nevertheless, despite extensive research and some promising outcomes from preclinical and clinical data, enthusiasm about Wnt inhibitors needs to be carefully evaluated. Firstly, the intricate nature of Wnt signaling and crosstalk with other signaling pathways are not fully elucidated. This lack of understanding most probably hinders the development of more efficacious therapeutic strategies. Secondly, we do not know whether the inhibition of Wnt signaling will activate resistance response mechanisms by other signaling pathways. Thirdly, the important role of Wnt signaling in physiological homeostasis and in bone remodeling, and the consequent adverse effects of its inhibition, cannot be dismissed. It is noteworthy to mention that toxicities originated from these treatments may vary dramatically depending on the targeted pathway component .In the case of FAK inhibitors, translation in the clinical setting is more advanced: four adenosine triphosphate (ATP)-competitive, small-molecule inhibitors of FAK , have been proven effective in several preclinical studies  and haveNF2) loss [NF2, although support for efficacy with this combination was limited [In a phase I study (NCT01138033), GSK2256098 showed clinical activity in patients with malignant mesothelioma, particularly those with merlin (encoded by F2) loss . With th limited . RecentlTwo studies have evaluated the effect of BI 853520 monotherapy in advanced or metastatic tumors (NCT01335269 and NCT01905111). These studies have demonstrated that BI 853520 has a manageable and acceptable safety profile and preliminary antitumor activity. However, as single-agent its activity was described as “modest”, therefore it has been proposed to explore this compound in combination with other agents for future studies .NF2 mutations (NCT04439331). Regarding patients with KRAS mutant lung cancer (NCT01951690), Defactinib monotherapy demonstrated modest clinical activity [VS-6062 was the first FAK inhibitor tested in clinical phase I trials (NCT00666926) but, despite promising results, it was discontinued due to nonlinear pharmacokinetics . Similaractivity . At presAltogether, it seems that the FAK inhibitors examined in different clinical trials have shown manageable toxicity profiles, some efficacy in delaying progression-free survival and maintaining stable disease in different advanced solid tumors; however, they did not produce objective clinical responses. Trials are currently directed to strengthen the efficacy by combining FAK inhibitors with cytotoxic chemotherapy, targeted therapy, or immunotherapy. In the future, prognostic markers could help to select patients who could benefit from FAK inhibitor treatment alone or in combination strategies .An important raising question is whether FAK inhibitors are effective as single agents. Since FAK is activated by so many binding partners and is interconnected with different signaling pathways, a combinatorial therapy seems the most promising approach . A numbeThe combinatorial therapy using FAK inhibitors with agents blocking other molecules or signaling pathways has already shown effectiveness over the use of single inhibitors in the preclinical setting. In a study of acute lymphoblastic leukemia, FAK downregulation enhanced the inhibitory effect of the mTOR inhibitor Rapamycin on cell growth in vitro, slowed the progression of leukemia in NOD/SCID mice in vivo, and prolonged the median survival time of the mice . Dual inmin/+ mice and reduced phospho-GSK3β and β-catenin intestinal levels, decreasing Wnt signaling [The many existing Wnt–FAK crosstalks in different cancers are complex, variable, and cell-type dependent. There is emerging in vitro and in vivo evidence that disrupting these crosstalks at multiple levels has therapeutic benefits. Pharmacological inhibition of FAK/PYK2 repressed adenoma formation in APCignaling . Co-targignaling . Indeed,ignaling . The inhignaling , suggestignaling .Nevertheless, despite the many examples of FAK–Wnt interactions described in the literature and summarized here, we are still far away from a comprehensive picture of this crosstalk, especially regarding its different cancer-specific dynamics. Some studies have started to develop mathematical models or network-based computational methods to quantify the signaling crosstalks in order to identify new therapeutic targets and evaluate the most effective drug target combinations ,151.In this review, we have highlighted the current knowledge on Wnt–FAK signaling crosstalks in different cancers , or a combinatorial approach targeting key molecules from both pathways , seem promising strategies. In fact, synergistic drug combinations are generally more specific to particular cellular contexts improving the therapeutically selectivity . The pro


Stromal cell‐derived factor‐1 (SDF‐1) actively directs endogenous cell homing. Exendin‐4 (EX‐4) promotes stem cell osteogenic differentiation. Studies revealed that EX‐4 strengthened SDF‐1‐mediated stem cell migration. However, the effects of SDF‐1 and EX‐4 on periodontal ligament stem cells (PDLSCs) and bone regeneration have not been investigated. In this study, we aimed to evaluate the effects of SDF‐1/EX‐4 cotherapy on PDLSCs in vitro and periodontal bone regeneration in vivo.Cell‐counting kit‐8 (CCK8), transwell assay, qRT‐PCR and western blot were used to determine the effects and mechanism of SDF‐1/EX‐4 cotherapy on PDLSCs in vitro. A rat periodontal bone defect model was developed to evaluate the effects of topical application of SDF‐1 and systemic injection of EX‐4 on endogenous cell recruitment, osteoclastogenesis and bone regeneration in vivo.+ cells and CD90+/CD34‐ stromal cells to the defects, enhanced early‐stage osteoclastogenesis and osteogenesis‐related markers expression in regenerated bone compared to control, SDF‐1 or EX‐4 in vivo.SDF‐1/EX‐4 cotherapy had additive effects on PDLSC proliferation, migration, alkaline phosphatase (ALP) activity, mineral deposition and osteogenesis‐related gene expression compared to SDF‐1 or EX‐4 in vitro. Pretreatment with ERK inhibitor U0126 blocked SDF‐1/EX‐4 cotherapy induced ERK signal activation and PDLSC proliferation. SDF‐1/EX‐4 cotherapy significantly promoted new bone formation, recruited more CXCR4SDF‐1/EX‐4 cotherapy synergistically regulated PDLSC activities, promoted periodontal bone formation, thereby providing a new strategy for periodontal bone regeneration. Locally delivered stromal cell‐derived factor‐1 (SDF‐1) combined with systemically applied Exendin‐4 (EX‐4) synergestically recruited endogenous stem cells towards the rat periodontal defects. Further, the cotherapy enhanced the proliferation and osteogenic differentiation of the engrafted stem cells in coordination, thus significantly facilitated periodontal bone regeneration. Afterwards, local application of SDF‐1 and systemic injection of EX‐4 were applied to a rat periodontal bone defect model. Endogenous cells recruitment, early osteolclasteogenesis and bone regeneration were evaluated to verify whether the cotherapy will provide a new strategic option for in situ periodontal bone regeneration.22.1The study protocol was approved by the School and Hospital of Stomatology, Shandong University (Protocol Number: GR201603), and written informed consent was obtained. Human PDLSCs were isolated according to our previous study.2.23 cells/well and cultured in basic media [Dulbecco's modified Eagle's medium (DMEM) with 10% foetal bovine serum (FBS)]. After 24 hours, cells were cultured in maintenance media (DMEM with 2% FBS) or maintenance media with SDF‐1, EX‐4, or SDF‐1+EX‐4 for 1, 3 or 5 days. Cell‐counting kit‐8  solution was added. The absorbance was detected by a microplate reader  at a wavelength of 450 nm. Experiments were performed in sextuplicate (N = 6).50 ng/mL SDF‐1 (Peprotech) and 10 nmol/L EX‐4 (R&D systems) were selected for the subsequent experiments according to our 2.34 cells/well and cultured in maintenance media. The lower plates were supplemented in 500 µL maintenance media with SDF‐1, EX‐4, or SDF‐1+EX‐4. 500 µL maintenance media served as a negative control (NC) and 500 µL basic media served as a positive control (PC). After 20 hours, cells that had migrated through the membrane were fixed with 4% paraformaldehyde (Sigma Aldrich) and stained with 0.1% crystal violet (Solarbio). Then cells were observed under a microscope and six randomly selected high‐power microscopic fields (×200) per filter were counted. Experiments were performed in triplicate (N = 3).The migratory effect of SDF‐1 and EX‐4 on PDLSCs was evaluated by an 8 µm transwell chamber (Corning). PDLSCs were seeded onto the upper chamber at a density of 5 × 102.45 cells/well and cultured in osteogenic inductive media , OM with SDF‐1, EX‐4, or SDF‐1+EX‐4. Experiments were performed in sextuplicate (N = 6).PDLSCs were seeded in 6‐well plates at a density of 2 × 102.4.1After 21‐day induction, PDLSCs were fixed with 4% paraformaldehyde and extracellular matrix calcification was estimated by 2% Alizarin Red S . 10% (w/v) cetylpyridinium chloride  and 10 mmol/L sodium phosphate solution were used to quantify the relative amount of calcium. The absorbance was measured at 562 nm wavelength.2.4.2After 7, 14 days induction, PDLSCs were lysed with 1% Triton‐X (Solarbio) for 30 minutes. Protein concentration of collected cell lysates was measured by BCA assay (Solarbio). Alkaline phosphatase (ALP) activity in these lysates was detected according to assay kit (Nanjing Jiancheng Bioengineering Institute). The absorbance was measured at 520 nm wavelength.2.4.3(−ΔΔCt) method. The sequences of the primers of GAPDH, ALP, osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OCN) were shown in Table After induction for 7, 14 and 21 days, total RNA was extracted with Trizol® (Takara). 1 μg mRNA was reverse‐transcribed to cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). Then qRT‐PCR was performed with SYBR® Premix Ex Taq II  on Light Cycler Roche 480 II Real‐Time PCR System (Roche). Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) primer was used as a housekeeping gene and data were analysed using the 22.55 cells/well and cultured in basic media. To examine the effect of SDF‐1 and EX‐4 cotherapy on extracellular signal‐regulated kinase (ERK), cells were cultured with basic media, SDF‐1, EX‐4, or SDF‐1+EX‐4 for 2 minutes. To determine whether the specific ERK inhibitor U0126 (MedChemExpress) influenced the ERK phosphorylation, cells were cultured with basic media, SDF‐1+EX‐4, SDF‐1+EX‐4+U0126 or U0126 for 2 minutes. Cells were then lysed with RIPA (Solarbio) containing 1% phosphatase inhibitor (CoWin Biosciences) and 1% protease inhibitor (Solarbio). The collected proteins were loaded onto 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% non‐fat dry milk and incubated with anti‐GAPDH antibody , anti‐total‐ERK antibody , or anti‐phospho‐ERK antibody  overnight at 4°C. The immunoreactive bands on membranes were incubated with horseradish peroxidase‐labelled secondary antibodies  for 1 hour and visualized with chemiluminescence system (Millpore) and analysed with image j 1.44 software (NIH). Experiments were performed in triplicate (N = 3).Cells were seeded in 6‐well plates at a density of 3 × 102.6A specific ERK inhibitor U0126 was used to clarify the role of ERK signalling pathway in SDF‐1+EX‐4 mediated PDLSC proliferation. Briefly, PDLSCs were seeded in 96‐well plates and cultured in basic media. After 24 hours, cells were cultured in maintenance media or maintenance media with SDF‐1+EX‐4, SDF‐1+EX‐4+U0126 or U0126 for 1, 3 or 5 days. Cell proliferation was quantified using a CCK8 kit as described above. Experiments were performed in sextuplicate (N = 6).2.72.7.1Medical collagen membranes (Zhenghai Biotechnology) were cut into small pieces, placed in 96‐well plates and cultured with 100 μL SDF‐1 (50 μg/mL) or phosphate‐buffered saline  according to our previous study.2.7.2The experimental procedure was approved by the Institutional Review Board, Hospital of Stomatology, Zunyi Medical University (Protocol Number: ZYKQ‐IRB‐KY‐2019‐002). Sixty 8‐week‐old adult Wistar rats  were used in this study. Periodontal defects were prepared on bilateral mandibles of each rat. The rats were randomly divided into two groups. Thirty rats intraperitoneal injected daily with 0.9% sodium chloride solution, the membrane loaded with PBS implanted into the left side defect was control group, while the membrane loaded with SDF‐1 implanted into the right side was SDF‐1 group. Thirty rats intraperitoneal injected daily with EX‐4, the membrane loaded with PBS implanted into the left side defect was EX‐4 group, while the membrane loaded with SDF‐1 implanted into the right side was SDF‐1+EX‐4 group. Six rats of each group were sacrificed respectively under general anaesthesia at 3 days, 1, 2, 4 and 8 weeks after surgery. Surgical procedures were performed as previously described.2.83d slicer software package .The samples of mandibles were scanned in three dimensions (3D) by a micro‐CT scanner (PerkinElmer). The effective voxel size was 50 μm in high‐resolution mode. The results were performed 3D reconstruction using Quantum GX micro‐CT software (PerkinElmer). Bone volume/tissue volume (BV/TV), bone surface area/tissue volume (BS/TV), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) were analysed by 2.9The bilateral mandibles of each rat were decalcified with 10% ethylenediaminetetraacetate , dehydrated with a series of graded ethanol solutions and embedded in paraffin wax. The embedded mandibles were cut in a buccal‐lingual direction to acquire transverse sections (5 μm thick). The prepared sections were stained with haematoxylin and eosin . The samples were observed under a BX53 microscope (Olympus Corporation) and measured with Image pro‐plus 6.0 Software (Media Cybernetics).2.10The mandible sections were incubated with antigen retrieval solution (Solarbio), blocked with goat serum (Solarbio) and incubated with primary antibodies at 4°C overnight: rabbit anti‐CXCR4 antibody , rabbit anit‐CD34 antibody  and mouse monoclonal anti‐CD90 antibody  for a double immunofluorescence staining of CD90/CD34. Goat anti‐rabbit IgG  or goat anti‐mouse IgG  was used as the secondary antibodies. Nuclei were visualized with 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride  and mounted. The images were photographed under a fluorescence microscope in the dark room.2.11+ cells was counted and measured by Image pro‐plus 6.0 software.Sections were stained with a leucocyte acid phosphatase kit (Solarbio) according to the manufacturer's instructions, nuclei were visualized with methyl green. The number of TRAP2.12image pro‐plus 6.0 software.The blocked sections were incubated with rabbit anti‐ALP antibody  or rabbit anti‐Collagen I (Col I) antibody  at 4°C overnight. Goat anti‐rabbit IgG H&L  was used as the secondary antibody. Immunoreactions were detected with 3,3′‐diaminobenzidine  solution. Nuclei were visualized with haematoxylin and mounted. The images were photographed under a microscope. The mean optical density (OD) of ALP and Col I staining was measured by 2.13graphpad prism software  and differences among more than two groups were analysed by one‐way or two‐way analysis of variance followed by Tukey's honestly significant difference comparison test. Value of P < .05 was defined as significant.All data were expressed as mean ± standard deviation (SD) and performed at least triplicates. Tests were analysed using 33.13.1.1P < .001; Figure P < .05). Meanwhile, EX‐4 presented no effect on PDLSC proliferation compared with control group (P > .05). The results showed that SDF‐1/EX‐4 cotherapy dramatically enhanced the proliferation of PDLSCs.SDF‐1/EX‐4 cotherapy significantly promoted the proliferation of PDLSCs compared with control, SDF‐1 or EX‐4 groups at day 3 and 5 . SDF‐1 or EX‐4 also promoted PDLSC migration compared with NC (P < .001), and the promotion effect of SDF‐1 on cell migration was stronger than that in EX‐4 group (P < .001). These results indicated that SDF‐1/EX‐4 cotherapy enhanced PDLSC migration.SDF‐1/EX‐4 cotherapy significantly enhanced the migration capacity of PDLSCs compared with NC, SDF‐1 or EX‐4 groups . SDF‐1+EX‐4 exhibited the maximal effect compared with the other three groups (P < .001). Unsurprisingly, SDF‐1, EX‐4 or SDF‐1+EX‐4 significantly upregulated ALP activity in comparison with OM at day 7 or 14 . Gene expression levels of ALP, OPN, BSP and OCN were significantly upregulated in SDF‐1+EX‐4 group when compared with the other three groups . Further, ERK signalling pathway was blocked with an inhibitor, U0126. SDF‐1/EX‐4 cotherapy promoted ERK phosphorylation was significantly inhibited by U0126 . Moreover, SDF‐1+EX‐4 group significantly increased the index of Tb.Th at week 2, 4 and 8 (P < .05), indicating that the newly formed bones were denser. Tb.Sp in all groups were in a downtrend during the healing process. SDF‐1+EX‐4 group had faster descent rate than the other three groups at week 1 and 2 (P < .05), illustrating that new bone formed and matured earlier.3D digital reconstructed micro‐CT images indicated SDF‐1+EX‐4 significantly promoted new bone formation compared with control, SDF‐1 or EX‐4 groups Figure . The new3.5P < .05). SDF‐1 or EX‐4 also formed more new bones than control group (P < .05). Almost no new bone formed in the defect area of control group, and a small amount of new bone formed on the edge of defects in the other three groups at week 1 (P < .01). Newly formed bones could be observed in control group at week 2, but the percentage of newly formed bones was significantly less than the other three groups (P < .05), and the difference continued to the experimental end point. At week 4, the defect area of SDF‐1+EX‐4 group was filled with new bones, and the newly formed bones were similar to normal bone tissues in SDF‐1+EX‐4 group at week 8, while less bone and immature bone trabeculae formed in control group.Periodontal bone repair was assessed by H&E staining and histomorphometric measurements at week 1, 2, 4 and 8 post‐surgery Figure . SDF‐1+E3.6+ cells in SDF‐1, EX‐4 or SDF‐1+EX‐4 groups was significantly larger than control group during the early stage of defect repair , and the promotion effect of SDF‐1 on CXCR4+ cell engraftment was stronger than that in EX‐4 group (P < .05). CXCR4+ cells decreased obviously at week 2, though the number of CXCR4+ cells in SDF‐1+EX‐4 group was still the largest (P < .01). At week 4, CXCR4+ cells in defect areas were hardly detected.The number of CXCR4+/CD34‐ cells in SDF‐1, EX‐4 or SDF‐1+EX‐4 groups significantly increased compared with control group . The number of CD90+/CD34‐ cells in SDF‐1+EX‐4 group was significantly larger than that in SDF‐1 or EX‐4 groups at three time points (P < .001). More CD90+/CD34− cells were observed in SDF‐1 group than EX‐4 group at day 3 or week 1 (P < .01). The number of CD90+/CD34− cells peaked at week 1 and decreased obviously at week 2. Unsurprisingly, few CD90+/CD34− cells were seen in the defect areas at week 4.During the early healing process , the number of CD90p Figure ; P < .013.7+ cells compared with control group at day 3 and week 1 . The number of TRAP+ cells in SDF‐1+EX‐4 group was two folds as much as that in SDF‐1 or EX‐4 groups at day 3 (P < .001). TRAP+ cells peaked at week 1, reduced obviously at week 2 and hardly detected at week 4.SDF‐1, EX‐4 or SDF‐1+EX‐4 significantly increased the number of TRAP1 Figure ; P < .053.8P < .01), while there was no significant difference between SDF‐1 and EX‐4 groups , while no significant difference was found between SDF‐1 and EX‐4 groups.ALP expression was significantly increased in SDF‐1, EX‐4 or SDF‐1+EX‐4 groups than that in control group at week 1 (4The purpose of this study was to evaluate the effect of cotherapy with a potent stromal cell homing factor SDF‐1 and an anti‐diabetic drug EX‐4 on periodontal bone regeneration. Firstly, we demonstrated that SDF‐1/EX‐4 cotherapy enhanced the proliferation, chemotaxis and osteogenic differentiation of PDLSCs in vitro. Secondly, periodontal bone regeneration was significantly promoted by topical application of SDF‐1 and systemic injection of EX‐4 in vivo.SDF‐1 triggered promotion effects on MSC proliferation and migration were mainly mediated through SDF‐1/CXCR4 signalling pathway.EX‐4, an anti‐diabetic agent, shares a 53% amino acid sequence homology with human GLP‐1.Several pathways have been reported to be involved in cell proliferation and migration, including ERK and PI3K/AKT pathways.ALP, BSP, OPN and OCN were significantly upregulated with SDF‐1/EX‐4 cotherapy at different stages in vitro. A previous report suggested that EX‐4 promoted osteogenic differentiation of BMSCs by regulating Wnt/β‐catenin signalling pathway.EX‐4 could directly promote MSC osteogenesis according to recent studies.The potential of SDF‐1/EX‐4 cotherapy on periodontal bone regeneration was evaluated by topical application of SDF‐1 and systemic injection of EX‐4 in a rat periodontal bone defect model. We previously reported that local delivery of SDF‐1 recruited host‐derived MSCs and promoted the quality and quantity of regenerated bones.+/CD34− cells were considered to be non‐haematopoietic stromal cells, which possess osteogenic differentiation potential. Our results showed that there was more CD90+/CD34− stromal cell mobilization after topical application of SDF‐1 alone. It is well known that SDF‐1 and CXCR4 interaction is responsible for recruiting MSCs and osteogenic progenitor cells together with the other types of cells to the injured sites.+ cells during the early stage of defect repair. SDF‐1 may enhance new bone formation by recruitment of endogenous cells.+/CD34‐ stromal cell and CXCR4+ cell migration to defect area. Moreover, EX‐4 significantly enhanced SDF‐1‐induced recruitment of the host cells. It's worth noting that SDF‐1 displayed more stronger potential to mobilize stem cells than EX‐4 in vitro and in vivo. However, the newly formed bones in SDF‐1 group were similar to EX‐4 group in periodontal defect in vivo. The findings suggest that regenerative strategies targeted at recruiting cells are not sufficient for successful periodontal bone regeneration. Orchestrating their differentiation into tissue‐forming cells in the next stage is extremely important.Adult stem cells expressing CD90, a cell surface marker of stem and progenitor cells, possess high potential to undergo osteogenic differentiation.Bone remodelling depends on the coordination of bone resorption by osteoclasts and bone matrix synthesis by osteoblasts, thus osteoclasts play a vital role in the process of bone repair.Further, rat periodontal defect model results indicated that SDF‐1 or EX‐4 alone enhanced periodontal bone formation. In addition, EX‐4 further enhanced SDF‐1 induced periodontal bone regeneration in vivo. There are some potential explanations for this synergetic effect. Firstly, MSCs and bone marrow‐derived osteoblast progenitor cells might be engrafted from the circulating blood towards the periodontal defects via SDF‐1 or EX‐4.In conclusion, cotherapy with 50 ng/mL SDF‐1 and 10 nmol/L EX‐4 enhanced the proliferation, chemotaxis and osteogenic differentiation of PDLSCs in vitro. Moreover, SDF‐1/EX‐4 cotherapy enhanced the recruitment of MSCs, induced early osteoclastogenesis, promoted the expression of osteogenic proteins in newly formed bone tissues and improved the quantity and quality of regenerated bones in vivo. Therefore, SDF‐1/EX‐4 cotherapy provides a new strategic option for in situ periodontal bone regeneration.The authors declare that there is no conflict of interests.QYL performed the experiments and wrote the manuscript. LQD performed the experiments and reviewed manuscript. RZ performed the experiments. WYK analysed the data and prepared figures. SHG designed the study and revised the manuscript.Supplementary MaterialClick here for additional data file.


It was found that the mechanical properties measured in compression mode on cylindrical specimens of unfilled and CaCO3-loaded epoxy were scarcely influenced by the kind of curing agent employed; only the compressive modulus was limitedly affected by this parameter. Referring to the behavior when aged in water, the CaCO3-filled epoxies displayed noticeable growths in modulus, small reductions in strength, and limited variations in strain, with a certain influence of the exposure time, especially when comparing the properties at the lowest time with those at medium–long times. On the basis of the results of statistical MANOVA analysis, it can be concluded that among the compositional factors , only the amount of CaCO3 filler significantly affects the compressive modulus. The effects of aging exposures to three non-saline aqueous environments on the compressive mechanical properties of a calcium carbonate-filled bisphenolic epoxy adhesive, cold-cured with the addition of two curing agents suitable for the cure at ambient temperature , were assessed. The amount of the added filler (CaCO Epoxy resins are commonly employed in an extensive variety of different applications as structural adhesives being characterized by a mix of advantageous outstanding properties, such as thermal stability and chemical resistance, high mechanical performance, and the capability to join very different  materials . TherefoThe introduction of the inorganic fillers is a common practice to adjust one or more properties of a polymeric matrix, not least to reduce its cost. The chemical nature and amount of added filler; its size, shape, and aspect ratio; its morphology; its degree of dispersion, and interactions with the polymer are the parameters mainly affecting the effects produced on the resin matrix, including its physical/mechanical properties and durability ,5,6. Although the use of nano-sized fillers has widely developed in recent years with the aim of providing the resins with new functions and improved performance ,9,10,11,3) is commonly added in different shapes  to polymers as the inorganic filler possessing many favorable features, such as ease of being incorporated in macromolecular materials, low environmental impact and toxicity, wide availability, and cheapness. This low-cost filler in micrometric size is able to improve the characteristics of epoxy coatings and paints , as well as the performance of epoxy-based adhesives and composites , to enhance the flashover voltage properties of epoxy intended for electrical applications [Calcium carbonate CaCO is commoications ,19,20,21Generally speaking, the transformation from a liquid resin to a hard, rigid, and resistant epoxy occurs by means of a chemical reaction, i.e., the cross-linking process, upon the addition to the resin of a wide range of curing agents, the more frequently used being polyfunctional amines and acid anhydrides. A distinctive advantage of the epoxy resins resides in their possibility to be cross-linked in any condition of temperature, ranging from ambient (even close to 0 °C) temperatures, as in the case of “cold-curing” epoxies applied on field as structural adhesives, to very high temperatures  for more demanding applications, for instance in the manufacture of high-performance composites based on heat-cured epoxy resin matrices. Depending on the application, and in turn on the temperature at which the resin must set and harden, a proper curing agent must be selected. The chemical nature and the composition of the epoxy/curing agent couple significantly affect the final properties and performance of the cured resins, as well as their long-term durability under service conditions. On the other hand, this latter characteristic also depends in a complex way on the environmental exposure and load regime ,23,24. TReferring to the environmental agents to which epoxy resins can be most frequently exposed, water is indubitably the more recurrent. Pure water can be present in liquid  or vapor (moisture) states; it is able to enter epoxies and to severely affect in reversible  or irreversible  ways the resin properties and load-bearing capabilities ,26,27,283 content, which is a phenomenon that is attributed to the more tortuous path that water encounters when it enters the resin in the presence of the micro-filler. The same studies also showed that the reductions in mechanical tensile properties (strength and modulus) due to plasticization of the resin were smaller than those observed for the same unfilled epoxy.It is reported that the calcium carbonate filler has a beneficial effect on the resistance of the epoxy resin to water ,32,33,343-loaded epoxy resins exposed to aqueous environments. On the other hand, the compressive properties play an important role when epoxies are used as structural adhesives in a wide variety of applications, applications in which the resins can be eventually exposed for prolonged times to the action of water .The mentioned studies, nor other studies to the best of our knowledge, did not take into consideration the compressive mechanical properties of a CaCO3 was assessed as a function of the kind of curing agent, the filler content, and the aging time. In the present study, the same experimental procedure was repeated employing a different resin commercially available as structural adhesive, i.e., a diglycidyl ether of bisphenol A epoxy modified with a polyester resin, cold-cured with the same curing agents, loaded with the same amounts of calcium carbonate micro-filler and aged to the same aging regimes. In fact, the aim of this paper was to establish if the interesting findings found in the previous study related to the effects of the filler on the mechanical behavior of a bisphenolic epoxy resin aged in water for prolonged times can be generalized for other cold-cured epoxy-based adhesives. Moreover, starting from cold-cured thermosetting adhesives displaying different mechanical properties, it is possible to identify the most suitable composition (in terms of resin/curing agent couple and filler content) for each application.In a previous study , the effThe resin object of the present study (trade name Epidian 57) was a diglycidyl ether of bisphenol A (DGEBA) epoxy modified with a polyester resin, supplied by Ciech-Sarzyna . The epoxide equivalent weight of Epidian 57 is about 250 g/mol; the resin, employed as adhesive cured at ambient temperature  to bond different materials , displays good mechanical properties, high shear and peel strengths, and a good resistance toward acids, except acetic and concentrated hydrochloric acids. Other properties and characteristics of Epidian 57 epoxy resin are reported in Epidian 57 epoxy resin was cold-cured upon the addition of two different curing agents, which are both suitable for the cure of epoxy resin at ambient temperature, namely TFF and Z-1. TFF is a Mannich base curing agent  displaying an amine number of 500–700 mg KOH/g. The TFF curing agent was selected for its fast reactivity with epoxy even at ambient temperature as well as being able to improve the chemical resistance and thus durability of the epoxy exposed to different environments. In The second curing agent selected to cure Epidian 57 resin at ambient temperature was a triethylenetetramine (TETA) with an amine number of 1100 mg KOH/g, again supplied by Ciech-Sarzyna Company . Some properties and characteristics of the Z-1 curing agent can be found in Two control (unfilled) formulations were produced: the cold-curing Epidian 57 epoxy with the Mannich base or TETA curing agents. The compositions (expressed in weight percentages and corresponding to the stoichiometric epoxy/amine molar ratio for each curing agent) of the reference formulations were Epidian 57:TFF 100:22 (indicated as E57/TFF/100:22 system) and Epidian 57:Z-1 100:10 (indicated as E57/Z-1/100:10 system), respectively.3 filler, i.e., 1, 2, or 3 g of the calcium carbonate per 100 g of epoxy resin; then, these epoxy mixtures were cold-cured upon the addition of two curing agents, i.e., TFF and Z-1. The compositions of the resulting epoxy compounds are reported in 3) filler, supplied in powder form by Carl Roth GmbH + Co KG  with the trade name E170, displayed a micrometric particle size, i.e., 0.87 ± 0.1 μm.Starting from Epidian 57 epoxy resin, modified formulations were produced by adding different amounts of CaCO3–epoxy systems can be found in a recently published paper [The detailed procedure to manufacture the CaCOed paper . After aed paper ,36,37,383 and cold-cured according to the procedure described in the previous paragraph, were exposed for prolonged times to different environments  in order to analyze the effects of aqueous media, differing for composition, on the mechanical behavior of the CaCO3–epoxy systems. The aim of the research was to assess if the presence of a substantially economic filler, such as calcium carbonate micro-particles, can improve the durability of any epoxy adhesive if exposed to a very common environment, such as non-saline water . In a previous paper, it was demonstrated that the presence of this micro-filler is able to mitigate the plasticization effect of water on the mechanical properties, tested in compression mode, of a cold-cured epoxy based adhesive [3-filled epoxy adhesives. The time of exposure was taken as an additional parameter of the study, the immersion in each aqueous environment being performed for 1, 6, and 10 months. An overview of the accomplished aging procedures is reported in The epoxy-based compounds, filled with different amounts of the CaCOadhesive : this re3, cold-cured as detailed in The epoxy-based compounds, unmodified or filled with different amounts of CaCOThe experimental results relative to the compressive modulus collected for each set of the composition/aging regime were subjected to a statistical analysis, eliminating the outermost results. To this aim, the ANOVA main effect analysis—the MANOVA analysis—and the Statistica 13.1 software were used. The statistical analysis was performed in two steps: the first step concerned the determination of normal distribution (at the significance level α = 0.05 using the Shapiro–Wilk test); in a second step, relevant statistical models and statistical tests were applied . In a further step, the Tukey test was used as a post hoc test to compare statistically significant factors.The effect of the addition of small amount of calcium carbonate micro-filler on the mechanical properties of a bis-phenolic epoxy resin, i.e., Epidian 57, cold-cured with the two selected curing agents, i.e., a Mannich base and a triethylenetetramine, was first assessed. The results of mechanical tests  performed on filled and unfilled epoxies in compression mode are presented in Analyzing first the effect due to the different curing agents selected for the cure of the epoxy at ambient temperature, it can be said that the compressive maximum strength b was sca3 filler to E57 resin brought about appreciable modifications to compressive mechanical properties: irrespective of the curing agent employed, a general increase in modulus along with a decrease in compressive strain at break were observed, the extent of the changes being influenced by the filler content. On the other hand, the compressive maximum strength remained substantially unaffected by the addition of calcium carbonate, regardless of the curing agent and the CaCO3 content.The addition of CaCO3 addition to E57 resin.By analyzing the results in depth, the addition of a small amount (2 g) of the micro-filler to the E57/TFF/100:22 system produced a marked growth in compressive modulus of over an order of magnitude a; even t3 addition to the epoxy resin under analysis is confirmed.Similarly, the addition of calcium carbonate to the E57/Z-1/100:10 system led to noticeable increases in compressive modulus, ranging an order of magnitude with the addition of 1 or 2 g of filler a; in cor3-filled epoxy was tested in tensile mode [3 with respect to the average modulus of an epoxy.The observed results are in good agreement with those found by adding the same amounts of the same filler to cold-cured Epidian 5 epoxy , i.e., aile mode ,31,33,343 filler did not affect significantly the compressive maximum strength of E57 epoxy, as can be seen from the data reported in As already mentioned, the addition of small amounts of CaCO3 filler produced growths in maximum compressive strength depending on the curing agent employed and on the amount of filler [Starting from a more resistant resin, i.e., Epidian 5 epoxy, the addition of up to 3 g of CaCOf filler . TherefoThe strain at break values measured on both E57/TFF/100:22 and E57/Z-1/100:10 systems containing 2 and 3 g of calcium carbonate were distinctively lower than those measured on the respective unfilled systems c. The tw3 filler. Moreover, these results, observed also when the CaCO3-filled epoxy resins are tested in tensile mode [These results are substantially in line with those presented in the previous mentioned study , where rile mode ,31,33,343 micro-filler, after prolonged exposures (up to 10 months) to three selected aqueous environments, reproducing realistic conditions involving immersion in unsalted water. The effects on the compressive mechanical characteristics of E57/TFF/100:22 system filled with calcium carbonate after immersion in demineralized, distilled, and spring water are presented in Then, the research was devoted to the assessment of the mechanical properties, tested in compression mode, of the epoxy adhesive cold-cured by the Mannich base curing agent and containing different amounts (from 1 to 3 g) of CaCO3, this property experienced an appreciable reduction after 1 month of immersion, even if the absolute value of the modulus is comparable to those measured on the other filled systems aged for the same time. Then, at longer immersion times, the modulus increased to a value close to that measured for unaged system. However, as already observed, a large standard deviation value was measured for the result relative to unaged E57/TFF/100:22/2CaCO3: this would explain the substantially different behavior of the modulus of this formulation upon immersion in demineralized water. Therefore, these data deserve additional investigation.Analyzing in detail the data recorded after different aging times in demineralized water a, this e3-filled Mannich base-cured E 57 epoxy systems experienced very small reductions with respect to the initial values  should be confirmed.In this case, the compressive modulus increased more gradually by increasing the immersion times a, achievAs regards the compressive maximum strength data, apart from the inevitable variations caused by the experimental measurements, the numerical data reported in 3-filled E 57 epoxy systems in The values of compressive strain at break reported for the CaCO3-filled E57/TFF/100:22 compounds immersed in spring water can be compared to the findings from an analogous investigation carried out on Mannich base-cured Epidian 5 epoxy filled with the same amounts of the same micro-filler and exposed for the same time spans in spring water [3-filled Epidian 5 epoxy measured in the previous study are very similar to what was experienced in the present study, with the exception of the larger reductions measured for compressive strain at break.Finally, the comparison of the results of mechanical tests performed in compression mode on CaCOng water . The sam3-filled Epidian 5 resin cold-cured with the Mannich base curing agent. On the other hand, if the adhesive must maintain the strain values substantially unchanged during the application of a compression load during its immersion in an aqueous environment, the choice should probably fall on one of the CaCO3-filled E57/TFF/100:22 systems.As already underlined, a distinctive advantage displayed by epoxy adhesives is represented by the possibility to select the type of resin, curing agent, and filler; the composition of the final system; and the curing conditions (in terms of temperature and time) in order to create the highest performing compound with respect to a specific application. Therefore, referring to an application for which frequent contact with a non-saline aqueous environment is expected, if the application requires the resin to withstand higher load resistance values (constantly greater than 100 MPa), the best choice would be CaCO3-filled E57/TFF/100:22 systems, aged in three aqueous environments and illustrated in the previous paragraph, was repeated by cold-curing the same filled epoxy resin, Epidian 57, with the TETA curing agent and subjecting the obtained systems to (compressive) mechanical tests after 1, 6, and 10 months of immersion in demineralized, distilled, and spring water, respectively. The results of these tests in terms of modulus, maximum strength, and strain at break are reported in The same investigation performed on the CaCO3 system seems to be out from this general trend: however, the very high standard deviation measured for this value could explain this discrepancy.The results recorded for the E57/Z-1/100:10 systems containing different amounts of calcium carbonate and immersed up to 10 months in demineralized water are first introduced . ReferriThe compressive maximum strength b experie3-filled E57/Z-1/100:10 systems after different time spans in demineralized water  than those of the other filled specimens was recorded, strong reductions of this parameter were measured as a consequence of the aqueous exposure. However, since this value appears out from the trend, as already underlined, it should be confirmed by further analysis.Substantial constant values of compressive strain at break were found for the CaCOed water c. Since 3-filled E57/TFF/100:22 specimens immersed for prolonged times in demineralized water. From the comparison, it can be concluded that the mechanical behavior observed for the filled Epidian 57 resin aged in demineralized water is almost independent on the curing agent used. In fact, this exposure caused appreciable increases in compressive modulus , i.e., noticeable increases in modulus, small reductions in the strength, and limited variations in the strain, can be regarded as general. There is a certain influence of the exposure time, especially when comparing the properties at the lowest time, i.e., 1 month, with those at medium–long times; the behavior does not depend on the type of water as long as it is not salty; it scarcely depends on the filler content ; the variations of the compression mechanical properties depend on the initial value of the property (such as the strain reductions observed for Epidian 5 and Epidian 57 resins).As a final conclusion, the mechanical behavior (measured in compression mode) observed for the cold-cured bis-phenolic CaCO3-filled E57/Z-1/100:10 specimens fractured during the mechanical tests are reported. The simple visual observation of the pictures revealed that the kind of damage experienced by the CaCO3-filled specimens mainly depends on the aging duration, while it apparently was not affected by the aqueous environment, thus further confirming what was revealed by the analysis of the experimental results. From the visual observation of the other compounds, i.e., those containing 2 and 3 g of the calcium carbonate filler, it was also possible to generalize that the time of exposure played a significant role on the kind of failure of the filled adhesives. In particular, it was noted that with increasing the aging time, the permanent deformation became more evident, especially after 6 months of exposure. At the longest aging time, i.e., 10 months, a cohesive failure of the micro-filled adhesive was generally recorded, with a clear appearance of some cracks and spalling.Finally, in 3-filler, cold-cured with two different curing agents, i.e., a Mannich base and a triethylenetetramine, both suitable to cure the resin at ambient temperature, can be altered as a consequence of the exposure to an aqueous environment for prolonged times. The findings found in the present study, summarized at the end of the previous paragraph, are discussed, also taking inspiration from the available literature.The investigation performed in the present study aimed at assessing how the mechanical behavior, tested in compression mode, of a commercial epoxy reinforced with the addition of small amounts of the CaCO3 filler to the epoxy resin has been shown to be effective in limiting the reductions in elastic modulus [As already underlined, the exposure to liquid water led to severe modifications of the properties and performance of epoxy resins. The addition of a proper filler to the resin can limit to a certain extent the detrimental plasticization effects caused by water ingress. When the mechanical properties of an unfilled epoxy are measured in tensile mode, both the elastic modulus and the maximum strength are reduced upon immersion in pure water; the addition of a CaCO modulus ,33,34. T modulus ,33. On t modulus ,33. This modulus ,28,40: a modulus .3 filler resulted in the growth of their tensile strain at break due to an increased ductile behavior caused by plasticization [3 micro-filler, which is able to hinder the absorption of the liquid in the resin [3 particles was found to additionally contribute to reduce the deformability of the resin [The exposure to an aqueous aging of epoxies added with a CaCOcization ,33,34. Ahe resin . Furtherhe resin . On the The analysis of the compressive modulus data, for which a greater spread of results was observed, was performed according to the ANOVA of the main effects, i.e., the MANOVA analysis. This is a multivariate analysis of variance used to assess the impact of different factors on the variable under study. This method was chosen since the variable under study, i.e., the modulus measured in compression mode, is influenced by several factors : therefore, it is important to analyze the influence of each factor on this variable.The adjustment of the results of empirical studies of the analyzed variables to the normal distribution was verified using the Shapiro–Wilk normality test (S-W);The distribution of the results obtained in the analyzed test variants was assessed, being consistent with the normal distribution.Verification of the assumption of homogeneity of variance was performed using Levene’s test.For the purpose of this statistical analysis:On the basis of the obtained results, the assumption of the normality of the distribution and homogeneity of variance was fulfilled (at a significance level α = 0.05). Then, the MANOVA analysis was performed for compressive modulus data collected for unaged and immersed specimens.p = 0.86644, there is no basis to reject the hypothesis that the “Type of aqueous environment” factor has no effect on compressive modulus;With the probability level of p = 0.000000, the hypothesis about no effect of the “Aging duration” factor should be rejected;With the probability level of p = 0.000000, the hypothesis that the “Amount of filler” factor has no effect should be rejected;With the probability level of p = 0.098346, there is no basis to reject the hypothesis of the lack of effect of the “Type of curing agent” factor.With the probability level of The results of the MANOVA statistical analysis applied to compressive modulus data allow concluding the following:3-filled epoxy adhesives, before and after their exposure to the three selected aqueous aging regimes, the analysis of variance demonstrated that both the aging duration and the amount of filler have a significant impact on the value of this mechanical characteristic; on the other hand, the type of aqueous environment did not significantly affect the modulus. Finally, this statistical analysis demonstrated that the type of curing agent influenced compressive modulus to a very limited extent.Therefore, when considering the compressive modulus data collected in the present study for CaCOAn appropriate post-hoc test was used to compare statistically significant factors. To this aim, the HSD Tukey test (“honestly significant difference” or “honest significant difference”) was employed, since it enables a homogeneous grouping of data with indication of significant differences. The Tukey test is used to determine if the relationship between two sets of data is statistically significant—that is, whether there is a strong chance that an observed numerical change in one value is causally related to an observed change in another value. The post-hoc test, the HSD Tukey test of homogeneous groups and significant differences, was performed. The obtained results are summarized in When analyzing the obtained results, it can be noticed that Tukey’s test separated three homogeneous groups. The samples aged for 6 and 10 months are in one homogeneous group, the remaining data belong to separate groups. This result would suggest that there are no significant differences between the samples aged for 6 months and for 10 months at the assumed significance level of α = 0.05. The obtained results were also confirmed by Tukey’s significant differences test: significant differences were not found between the groups of samples aged for 6 and 10 months, the obtained p values  are greater than the adopted one, i.e., 0.05.When the compressive modulus data were grouped according to the amount of filler, Tukey’s test distinguished two homogeneous groups. The modulus of pristine (unfilled) epoxy compounds was significantly different from the same property measured on the filled epoxy compounds, which was also confirmed by Tukey’s test of significant differences. The closer the value in this table (shown in black) to 1, i.e., it is not significantly different, the closer the results in these groups are to each other.The obtained results quantitatively confirmed what was already observed during the analysis of the results of compressive tests performed on calcium carbonate-filled epoxy compounds cured with two different curing agents and exposed for different times to different aqueous regimes.3 filler significantly affects the compressive modulus. The reproduced aging regimes, on the other hand, did not affect at all the compressive modulus, while the durability tests durations noticeably did.On the basis of the presented MANOVA statistical analysis, it should be emphasized that among the compositional factors , only the amount of CaCO3 filler, the epoxy adhesive, cured with two different curing agents suitable for the cure at ambient temperature, experienced noticeable increases in modulus upon immersion in different non-saline aqueous environments; the strain was somehow reduced, while the compressive strength was only barely affected by these aging regimes. The effect of the kind of the non-saline aqueous environment analyzed  was substantially negligible, as also confirmed by the MANOVA statistical analysis performed on the compressive modulus results. Referring to the exposure times, the mechanical characteristics were affected by this parameter only when comparing the properties at the lowest immersion time with those at medium-long times.The results of this study allowed confirming what was already been observed in a previous work, namely the effectiveness of adding small quantities of an inorganic inexpensive macro-filler, such as a calcium carbonate, to counteract the detrimental effects of aging in water on compressive properties of cold-curing epoxy adhesives. It was observed that with the addition of low amounts (from 1 to 3 g per 100 g of resin) of the CaCO3-filled epoxy, even after the exposure to an aqueous environment, was able to maintain the modulus much greater than, and the maximum strength very close to, the same characteristics measured on the unaged control adhesives.Analyzing the compressive mechanical properties relative to the unaged filled and the pristine epoxy adhesives, it was concluded that the micro-filler was able to affect to a great extent the compressive modulus, which increased with respect to the unfilled resin, and the strain values, which experienced noticeable reductions, irrespective to the curing agent used; once again, the strength of the epoxy was scarcely influenced by the addition of the filler. The mechanical characterization showed that the filler content had a marginal effect on the results. In any case, the CaCO