Helicobacter pylori infection. In all, 1225 incident stomach cancers (78% noncardia) were diagnosed over 7 094 637 person‐years; 984 in 382 957 study participants with lifetime alcohol intake data (5 455 507 person‐years). Although lifetime alcohol intake was not associated with overall stomach cancer risk, we observed a weak positive association with noncardia cancer , with a HR of 1.50 (95% CI: 1.08‐2.09) for ≥60 g/d compared to 0.1 to 4.9 g/d. A weak inverse association with cardia cancer  was also observed. HRs of 1.48 (95% CI: 1.10‐1.99) for noncardia and 0.51 (95% CI: 0.26‐1.03) for cardia cancer were observed for a life course trajectory characterized by heavy decreasing intake compared to light stable intake (Phomogeneity = .02). These associations did not differ appreciably by smoking or H pylori infection status. Limiting alcohol use during lifetime, particularly avoiding heavy use during early adulthood, might help prevent noncardia stomach cancer. Heterogeneous associations observed for cardia and noncardia cancers may indicate etiologic differences.Alcohol consumption is causally linked to several cancers but the evidence for stomach cancer is inconclusive. In our study, the association between long‐term alcohol intake and risk of stomach cancer and its subtypes was evaluated. We performed a pooled analysis of data collected at baseline from 491 714 participants in the European Prospective Investigation into Cancer and Nutrition and the Melbourne Collaborative Cohort Study. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated for incident stomach cancer in relation to lifetime alcohol intake and group‐based life course intake trajectories, adjusted for potential confounders including  What's new?Helicobacter pylori infection. A weak inverse association was observed with cardia cancer, suggesting that there may be etiologic differences between non‐cardia vs. cardia subtypes.Alcohol consumption has been causally linked to several cancers, but results for stomach cancer have been inconclusive. In this large, international study, the authors found a positive association between long‐term, heavy drinking and non‐cardia stomach cancer. This association persisted when data were adjusted for potentially confounding factors such as smoking, body mass index, and  CIconfidence intervalEPICEuropean Prospective Investigation into Cancer and NutritionHRhazard ratioICD‐O‐3International Classification of Diseases for OncologyMCCSMelbourne Collaborative Cohort StudyVCRVictorian Cancer Registry1Helicobacter pylori and Epstein‐Barr virusprobable causes of stomach cancer.Stomach cancer is the fifth most common cancer with an estimated 1 033 701 incident cases  worldwide in 2018.Alcohol drinking caused an estimated 3 million deaths globally  and an estimated 400 000 deaths from cancer  in 2016.In our study, we estimated associations for baseline and lifetime alcohol use with risk of stomach cancer and its subtypes, using retrospective information on consumption at various ages before recruitment in two large prospective studies.2A pooled analysis of 491 714 participants in the European Prospective Investigation into Cancer and Nutrition (EPIC) and the Melbourne Collaborative Cohort Study (MCCS) was conducted.2.12.1.1EPIC is a multicenter prospective cohort study of 521 324 participants (70.6% women), mostly of Caucasian descent, aged 35 to 70 years when enrolled, predominantly during 1992to 1998, designed to investigate the relationship among dietary habits, nutritional status and various lifestyle/environmental factors and cancer incidence.2.1.2H pylori status was determined in nested case‐control studies on stomach cancerH pylori‐seropositivity have been published previously.Diet over the 12 months before enrolment was measured by validated country‐specific dietary questionnaires2.1.3Alcohol intake at baseline was estimated from validated dietary questionnaires where participants reported the number of standard glasses of wine, beer, cider, sweet liquor, distilled spirits and fortified wines they consumed daily or weekly during the 12 months before recruitment.2.1.4Cases were identified from population cancer registries except in France, Germany, Greece and Naples , where a combination of different methods, including accessing health insurance records, hospital‐based cancer and pathology registries and active follow‐up (participant tracking), were used. Incident primary invasive stomach cancer cases were defined as those with code C16 according to the 10th revision of the International Classification of Diseases . Histologic type2.22.2.1The MCCS is a prospective cohort study of 41 513 people , all of white European descent, recruited during 1990to 1994 in Melbourne, Australia.2.2.2H pylori status was determined in nested case‐control studies of stomach cancerH pylori‐seropositivity have been published previously for MCCSStructured interview schedules were used to obtain information on potential risk factors including age, sex, country of birth, education, previous medical conditions and lifestyle behaviors . A 121‐item food frequency questionnaire was used to collect dietary information.2.2.3Participants were asked at baseline if they had ever drunk at least 12 alcoholic drinks in a year. Those who had (“nonlifetime abstainers”) were then asked about their usual frequency of consumption and usual quantity consumed per drinking occasion for beer, wine and spirits separately during 10‐year age periods commencing at age 20, up to the decade of their age at baseline attendance. Usual intake within each age period in grams per day for each beverage type was calculated by multiplying intake frequency by quantity and standard amount of alcohol per container using Australian food composition tables.2.2.4Cases and vital status were ascertained through the Victorian Cancer Registry (VCR), the Victorian Registry of Births, Deaths and Marriages, the National Death Index and the Australian Cancer Database. The outcome was defined as a histopathological diagnosis of primary invasive adenocarcinoma of the stomach, coded following the 3rd Revision of the International Classification of Diseases for Oncology (ICD‐O‐3) as C16. Tumor histopathology and anatomical site were obtained from the VCR.2.3The association between alcohol intake and stomach cancer was examined after pooling EPIC and MCCS data. Cox regression2), intake of total red and processed meat (g/d), fruit intake (g/d) and total energy from food not including alcoholic beverages   and for intake categories derived using the following cut‐points: nondrinkers, 0.1‐4.9, 5‐14.9, 15‐29.9, 30‐59.9, ≥60 g/d for total alcohol; nondrinkers, 0.1‐4.9, 5‐14.9, 15‐29.9, ≥30 g/d for beer and wine; and nondrinkers, 0.1‐4.9, 5‐14.9, ≥15 g/d for spirits. Consistently throughout our study, the 0.1‐4.9 g/d category  was used as the reference group. Nearly 78% of the eligible study participants (n = 382 957) were included in analyses for lifetime alcohol intake  and histologic subtypes  of stomach cancer, Cox regression models were fit in competing risks analysis.H pylori infection status and confounding due to it were evaluated for noncardia cancer using pooled data from EPICA dose‐response relationship between lifetime alcohol intake and stomach cancer incidence was examined by comparing models that included alcohol as a linear term and with restricted cubic splines , with upper intake capped at 100 g/d.Sensitivity analyses were performed (a) excluding the first 2 years of follow‐up, (b) without adjustment for body mass index and dietary covariates and (c) by estimating study‐specific HRs and pooling study‐specific results using random effects models. Each model was examined for outliers and influential points. Nested models were compared using likelihood ratio tests. Tests based on Schoenfeld residuals showed no departure from the proportional hazard assumptions. All statistical tests were two‐sided, and statistical analyses were performed using Stata 16.1 .3Characteristics of the 491 714 participants are described in Table About one‐third of participants was either lifetime abstainers or drank less than 5 g/d, and 2.2% reported a lifetime intake of ≥60 g/d Table . Althoug3.1P = .94). In analyses for subsites of the stomach, a 10 g/d increment in lifetime alcohol intake was weakly associated with increased incidence of noncardia cancer  and showed a weak inverse association with cancer of the gastric cardia  (Phomogeneity < .01) (Table Phomogeneity = .03) , and the inverse association with cardia cancer was observed in men only (Pinteraction = .02)  Table . The HR 1) Table . Using i3) Table . When stPhomogeneity = .97)  and overlapping/unspecified stomach cancers (C16.8 and C16.9) ; the corresponding HR for cancer of the gastric cardia was 0.51 (95% CI: 0.26‐1.03) (Phomogeneity = .02)   Table . There w3.4Phomogeneity = .02) , 0.93 (95% CI: 0.86‐1.00) and 1.03 (95% CI: 1.00‐1.06), thus displaying very similar estimates (and corresponding CIs) to aggregate‐level data. Associations between alcohol intake and overall and subtypes of stomach cancer incidence did not change appreciably when the first 2 years of follow‐up were excluded  or unspecified (C16.9) for site,Study strengths include the prospective design, comprehensive assessment of alcohol intake over the life course based on intakes at different ages, and over 15 years of follow‐up on average. Additionally, the large study size with nearly half‐a‐million study participants and over one thousand incident stomach cancers enabled the examination of associations for subtypes and by sex, cigarette smoking and body fatness and we were also able to study individual alcoholic beverage types as well as heavy consumption. Information on H pylori infection status in contrast with previous findingsAlcohol intake is classified as a probable cause of stomach cancer based on evidence for an association for intakes of 45 g/d or more, compared to abstention, mostly using data on alcohol intake at recruitment from case‐control studies.No previous observational study examined drinking trajectories in relation to stomach cancer. The association of noncardia cancer with heavy drinking during early adulthood using trajectories and the association with lifetime alcohol intake in our study are suggestive of early initiation and chronic progression of carcinogenesis linked to alcohol and its metabolites.Cancers of the gastric cardia and noncardia differ substantially in their patterns of incidence and etiology.H pylori infection status. Limiting alcohol use during lifetime, particularly avoiding heavy use during early adulthood, might help prevent noncardia stomach cancer.In conclusion, lifetime alcohol intake was associated with increased risk of noncardia stomach cancer, independent of smoking, body mass index and www.icmje.org/coi_disclosure.pdf and declare: no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; and no other relationships or activities that could appear to have influenced the submitted work.All authors have completed the ICMJE uniform disclosure form at Where authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization.The ethical review boards of the International Agency for Research on Cancer and all local institutions where participants had been recruited gave approval for the study. All participants gave written informed consent. The MCCS study protocol was approved by the Cancer Council Victoria Human Research Ethics Committee. Participants gave written informed consent to participate and for investigators to obtain access to their medical records.Appendix S1: Supporting InformationClick here for additional data file.


To systematically review and analyse the associations between fat and muscle mass measures with overall survival in men with prostate cancer.A systematic search was conducted in CINAHL, Cochrane Library, EMBASE, PubMed, and Web of Science databases from inception to December 2020, while abstracts from the American Society of Clinical Oncology (ASCO), Clinical Oncology Society of Australia (COSA), and the American College of Sports Medicine (ACSM) conferences were searched from 2014 to 2020. Eligible articles examined the association of body composition measures, such as fat mass , subcutaneous adipose tissue (SAT), and VAT/SAT) and muscle mass measures, with overall survival in prostate cancer patients at any treatment stage. The primary endpoint was overall survival. Random-effect meta-analysis was conducted for studies reporting multivariable or univariable analysis assessing the associations of fat mass measures  and muscle mass measures with overall survival.p = 0.888) and VAT  were not significantly associated with overall survival, while higher subcutaneous adipose tissue levels were associated with higher survival . Greater mortality risk was found in patients with localised  and advanced disease  presenting with low levels of muscle mass compared to those presenting with high levels.Sixteen cohort studies that comprised 4807 men with prostate cancer were included. Total adiposity (hazard ratio (HR) 0.98, 95% CI: 0.75–1.28, These results indicate that although overall adiposity should be cautiously interpreted in regards to survival, high muscle mass and SAT, and low VAT/SAT ratio values are associated with overall survival in men with prostate cancer. Prostate cancer is one of the most prevalent cancers worldwide, accounting for one in five new cancer cases in men . Among t−2) and a 23% increased risk for all-cause mortality [−2 with a 20% increased risk of prostate cancer-specific mortality [Obesity is a potential predictor of mortality in men with prostate cancer , 9, affeortality  were reportality , 15, or ortality . This aportality , 18, masortality , 20. Furortality , 21–23, ortality , 24, 25.As a result, we investigated in this systematic review the role of body composition on overall survival in men with prostate cancer, analysing the associations of low muscle mass and high fat mass as prognostic factors. In addition, a range of possible clinical  and methodological  variables that may affect the associations of body composition with overall survival were examined by subgroup analyses.A systematic search was conducted in the following electronic databases: CINAHL, Cochrane Library, EMBASE, PubMed and Web of Science from inception to December 2020. The search strategy is presented in the Supplementary eAppendix This review included published articles and conference abstracts  of studiData extraction was performed via a standardised form. Clinical and methodological information were extracted from the included studies such as cancer stage and treatment, number of participants at baseline, geographical region, age and BMI at baseline, fat and muscle mass assessments , follow-up period, HR for overall survival with their associated dispersion values such as 95% CI or standard errors (SE) from univariable and multivariable analyses, when available, and the number of covariates included in the multivariable models.The study quality assessment was evaluated according to the Newcastle-Ottawa Quality Assessment Scale (NOS) for cohort studies . The NOSp value of ≤0.05 was considered statistically significant. Forest plots were generated to present the results for multivariable and univariable analysis of fat and muscle mass. Heterogeneity between studies was assessed by using the I2 statistic and the p value from χ2-based Cochran’s Q test with a high heterogeneity defined by a threshold p value of 0.1 or I2 value greater than 50% [Data from the associations of body composition with overall survival such as HR and their associated dispersion values were pooled using inverse variance random-effects models. These values were extracted from univariable and multivariable models and log-transformed to be included in further analyses. Analyses were conducted for studies reporting multivariable or univariable analysis and subgroup analyses were provided for the following: (1) removing outliers; (2) for prostate cancer subgroups (stage or phase of treatment); (3) previously defined or median cut-off values for muscle mass outcomes; (4) specific depots of fat and (5) studies using BMI as a covariate or not in the multivariable models. A than 50% . We examthan 50% . AnalyseOf the 805 retrieved studies, 514 potential records were retained for screening after duplicate removals. Of these, 373 were excluded due to their irrelevance to the research question and 141 articles were deemed eligible and undertaken for review Fig. . A total−2 (IQR: 24.3–28.7) participated in the included studies. All studies except one derived fat and muscle mass measures from CT scans [n = 11) included advanced prostate cancer patients  [n = 9 for both), followed by surgery [n = 7 for both), and novel hormonal agents such as abiraterone and enzalutamide (n = 1) [The characteristics of the individual studies are presented in Tables −2 IQR: .3–28.7 pCT scans , 34–45. −2 IQR: .3–28.7 patients) , 41–45, atients) , 36–43 aatients) , 40–44 , while VAT and SAT  and SAT and VAT/SAT  were significantly different  or for studies controlling for BMI . In the univariable analysis, a 23% survival advantage was found after removing the study of Stangl-Kremser et al. [p = 0.061–0.438; Table p = 0.146; Supplementary eFig. Eight studies , 43–45 c2 m−2 , 58.7 cm2 [2 m−2 , 59.4 cm2 m−2 [2  and 287.−2 [2 [2 ) [24, 35−2 [2 [2 , 43–45, 2 m−2 [2 ) [24, 352 m−2 [2 , 43, 45,2 m−2 [2 ) [34, 442 m−2 [2 . Given t2 m−2 [2 , 43, 45 2 m−2 [2 m−2 [2 m−2 [2 m−2 [2 m−2 [2 m−2 [2 m−2 [2 m−2 [I2 = 58%. The results were maintained in the subgroup analyses  except for studies controlling for BMI, which approached statistical significance . Similarly, results were similar in univariable model analyses . No publication bias was found  [16, 242 [2 m−2 , 5.7 cm22 [2 m−2  and 7.5 2 [2 m−2 ) [25, 362 [2 m−2 , one stu2 [2 m−2  or skeleIn this review we examined the role of fat and muscle mass on survival in men with prostate cancer. The main findings of our study were: (1) although overall fat mass was not a prognostic factor in men with prostate cancer, high levels of subcutaneous fat and low levels of VAT/SAT were associated with a 32% and 50% survival advantage, respectively, in patients at advanced stages of the disease; and (2) patients presenting with low muscle mass levels are at ~50% increased risk of mortality compared to those presenting with high levels regardless of the cancer stage or methodological characteristics. These results are clinically relevant and indicate the importance of muscle mass in particular during the course of therapy given the substantial impact on overall survival of patients with prostate cancer.Although obesity and the resulting metabolic environment are deemed important factors for biochemical recurrence, metastatic disease and mortality in men with prostate cancer , 9, our Contrary to the results regarding total adiposity, a high level of muscle mass was associated with improved overall survival in prostate cancer patients regardless of treatment stage or methodological characteristics. This may be related to the numerous benefits of muscle mass on metabolic health such as regulating and mobilising natural killer cells into the tumour, or even altering other biomarkers associated with the tumour biology , 61. In n = 16) with up to 4807 prostate cancer patients included; (2) the assessment of both univariable and multivariable models; and (3) subgroup analyses based on different clinical and methodological characteristics. However, there are also some limitations which are worthy of comment. First, only cohort studies undertaking retrospective analyses were included in our review and this precludes determining causality of body composition changes such as increase in muscle mass and reduction in fat mass on overall survival. Future prospective analyses are necessary to improve current knowledge by indicating if interventions targeting fat and muscle mass are able to improve overall and disease-specific survival as well as recurrence in men with prostate cancer. In addition, researchers used different definitions for sarcopenia such as the skeletal muscle index, psoas muscle or median values, while no definition for adiposity measures was used, leading to high heterogeneity within the models. Sarcopenia has become a topic of great interest in oncology [The strengths of the present study are: (1) a relatively large number of studies (oncology ; howeveroncology –43, 45, As far as we are aware, the present systematic review and meta-analysis is the first to examine the prognostic value of fat and muscle mass in men with prostate cancer. In summary, increased levels of muscle mass and SAT and reduced VAT/SAT ratio rather than overall adiposity are important prognostic factors in men with prostate cancer, even when controlling for multiple confounding factors. Furthermore, we provide rationale for future prospective analyses investigating the impact of sarcopenia and changes in muscle mass during cancer treatment on prostate cancer outcomes, as well as the investigation of strategies such as exercise and nutritional interventions to improve survival in this population.Supplementary material file


It is uncertain whether cardiac resynchronization therapy with a defibrillator (CRT‐D) provides better survival benefits than a CRT‐pacemaker (CRT‐P) in heart failure patients with a reduced ejection fraction  treated with contemporary HF therapy.p = 0.03), but the gender distribution did not differ . The VA events during a median follow‐up of 3.5‐years (interquartile range [IQR]:1.6–5.5) and their predictors were analyzed.We retrospectively analyzed the ventricular arrhythmia  events in HFrEF patients who underwent CRT without a prior history of VAs or aborted sudden cardiac death before the CRT implantation. Between January/2010 and December/2020, a CRT device was implanted in 79 HFrEF patients . CRT‐D and CRT‐P devices were implanted in 50 and 29 patients, respectively, at each physician's discretion. CRT‐Ds were indicated in younger patients than were CRT‐Ps . The VA event rate was significantly lower in patients without a prior non‐sustained ventricular tachycardia  and females . Of note, no female patients without a prior history of NSVT experienced VA events.VA events occurred in 9 patients with CRT‐Ds (18%) and one with a CRT‐P (3%, HFrEF CRT candidates without a prior history of NSVT and females may obtain less benefit from a primary preventive defibrillator indication. It enrolled patients eligible to receive a CRT based on the current guideline indications.2.2At baseline and all the follow‐ups, the LVEF, LV end‐systolic volume (LVESV), LV end‐diastolic volume, mitral regurgitation, and interventricular dyssynchrony were assessed by echocardiography. Cardiac volumes were estimated using the modified Simpson method or Teichholz method. When only a visual assessment was available, the lower number of the LVEF range was used for the analysis.2.3The VA events during a median follow‐up of 3.5 years (IQR:1.6–5.5) and their predictors were analyzed. VA events were defined as sustained VAs, which needed anti‐tachycardia pacing or shock therapy to terminate. Non‐sustained ventricular tachycardia (NSVT) was defined as ≧3 consecutive ventricular premature contractions at a rate of >120 bpm and lasting <30 s. A CRT volume‐responder was defined as those with an LV end‐systolic volume reduction of >15%. The data for the follow‐up assessment were obtained from the pacemaker clinic database and electronic patient records.Outcomes of interest included the mortality and cardiac hospitalizations as tracked by the electronic patient records and databases. Cardiac hospitalizations included hospital admissions for any cardiac related event as well as unexplained syncope. Clinical follow‐up visits and device interrogations were scheduled at 1, 3, 6, and 12 months after the procedure, with remote device monitoring when possible.2.4p < 0.10 in the univariate analysis were included in the multivariate analysis, and the hazard ratios (HR) and 95% confidence interval (CI) were calculated. A value of p < 0.05 was considered statistically significant. All statistical analyses were performed using EZR on the R commander, version 1.36, software.Continuous variables are presented as either means (±standard deviation [SD]) or medians (with interquartile range [IQR]) and categorical variables as numbers and percentages (%). Differences in the continuous variables between the two groups were assessed by an unpaired t‐test or Mann–Whitney test. Categorical variables were compared through a Fisher exact test. The Kaplan–Meier method was used to estimate the survival function in patients treated with CRT. The follow‐up period from the last procedure was calculated with the median value and interquartile range. At first, to assess the clinical predictors of VT events and survival from all‐cause death, a univariate Cox proportional analysis was performed. Sequentially, variables with a 33.1During the study period, 79 patients underwent CRT implantations, and CRT‐D and CRT‐P devices were implanted in 50 and 29 patients, respectively, at the physicians' discretion Figure . The bas3.23.2.1During a median follow‐up period of 3.5 years , 10 (13%) of 79 patients experienced VA events. However, the VA events did not significantly differ between the patients with an EF < 30% and those with an EF = 30–35% (HR = 0.51 [95%CI:0.14–1.82]) or CRT volume responders and non‐responders (HR = 0.41 [95%CI:0.12–1.43]) (Table 3.2.2p < 0.01) was associated with fewer VA events. On the other hand, ICM  was not an independent predictor of VA events. A Cox proportional model was used for the multivariate analysis, and variates with a p < 0.10 in the univariate analysis were included in the model. A female gender  and no prior history of NSVT  were independent predictors of fewer VA events in the multivariate analysis. Furthermore, all female patients without a prior history of NSVT experienced no VA events.Table 3.2.3p = 0.049), and no prior history of NSVT  and the eGFR  were associated with a decreased risk of all‐cause mortality in the multivariate analysis.Thirty‐two patients (41%) died during the follow‐up. During a median follow‐up period of 3.5 years, the cumulative incidence of deaths due to HF was 23.1% (95% CI 13.2 to 34.7); the incidences of noncardiac death and SCD were 16.7% (95% CI 8.7 to 27.0) and 2.5% (95% CI 0.5 to 7.9), respectively , 13% of the patients who were implanted with a CRT‐D/CRT‐P for primary prevention experienced VA events.A “female gender” and “no prior history of NSVT” were independent predictors of fewer VA events in the multivariate analysis.There was no difference in the VA event and mortality rates between the CRT‐D and CRT‐P groups.We evaluated the VA events in HFrEF patients who were implanted with a CRT‐D or CRT‐P for primary prevention. The main findings of our study were:4.2In the present study, VA events occurred significantly less often in women than men. However, there was no difference in the all‐cause death rates between men and women. There was a potentially lesser benefit of primary prevention ICDs in women, because SCD may have a smaller impact on the total mortality in women than in men. A previous study showed that women receive significantly fewer appropriate ICD shocks.How sex differences translate into clinical arrhythmias and possibly appropriate ICD shocks has not been fully studied. There were sex differences in the electrophysiological properties such as repolarization, calcium handling, autonomic modulation, and ion channels.4.3Identifying patients at high risk of SCD remains a challenge. Many studies have investigated the predictive value of NSVT for SCD, but the results are ambiguous. Some studies have shown that NSVT does not increase the risk of SCD and all‐cause mortality4.4ClinicalTrials.gov number NCT03494933) that will randomize HFrEF and a CRT indication to either a CRT‐D or CRT‐P implantation. The randomized RESET‐CRT trial will provide urgently needed evidence in this debated field.The question of whether patients with HFrEF should receive a CRT‐D or CRT‐P is one of the major dilemmas in routine clinical practice in cardiovascular medicine. Especially given the physique and BMI of older Asian women, doctors are hesitant to implant physically large devices, like a CRT‐D or ICD. In this real‐world data, only 1 patient with a CRT‐P experienced SCD. The result of the present study demonstrated that our choice of CRT devices would have been acceptable. Furthermore, “no previous history of NSVT” and a “female gender” may be factors that are less likely to cause events after a CRT implantation. Nevertheless, the latest Japanese data from a sub‐analysis of the Nippon Storm study, demonstrated that the rate of appropriate ICD therapy is commonly applied at comparable rates for either primary or secondary prophylaxis in ICM patients with HFrEF.In clinical practice, the GDMT for HFrEF has improved dramatically. Patients without contraindications appear to gain the most benefit from a combined treatment with the ‘fantastic four’: an angiotensin receptor/neprilysin inhibitor (ARNI), beta‐blocker, MRA, and sodium–glucose co‐transporter 2 (SGLT2) inhibitor.4.5First, this study was a single‐center retrospective observational analysis and the number of study patients was relatively small. However, the observation period was considered to be adequate to analyze the ventricular arrhythmia events after CRT implantations . Additional multicenter, randomized trials should be performed to obtain a more objective assessment. Second, this study included patients with ICM and non‐ICM, and the non‐ICM consisted of a non‐uniform etiology. Moreover, sex differences in the distribution of ICM are also a limitation of the present study. Nevertheless, all the consecutive patients who underwent CRT implantations at our hospital met the inclusion criteria (LVEF<35 and primary prevention of SCD), which would have reduced the potential selection bias. Third, there was a possibility that the differences in the baseline characteristics between the CRT‐D and CRT‐P groups affected the incidence of VA events and the mortality rate after the CRT implantation. Fourth, whether to implant a CRT‐D or CRT‐P was left to the physicians' discretion in the present study. Further prospective studies are needed to elucidate this issue. Fifth, the detection and therapy programming in the tachycardia zones depended on each patient. Sixth, there was no uniformity in how and when the history of NSVT in each patient was determined. As the previous study showed, frequent monitoring would be more reliable to identify the importance of NSVT before a device implantation.5In a contemporary real‐world cohort of patients with HFrEF treated with CRT and with an indication for a primary prevention ICD, the HFrEF CRT candidates without a prior history of NSVT or that were female may have gained less benefit from a primary prevention defibrillator indication. These data may support the decision‐making as to whether to implant a CRT‐D or CRT‐P in HFrEF patients for primary prevention of SCD.Approval of the research protocol: The study was performed in compliance with the principles outlined in the Declaration of Helsinki and approved by the ethics committee of Kobe University Hospital .The patients consented to the use of their anonymized clinical data for research purposes by the opt‐out method.N/A.N/A.Toshihiro Nakamura: clinical practice/data sampling/drafting article. Koji Fukuzawa: concept/design/data analysis/interpretation/drafting article. Kunihiko Kiuchi: data collection/statics. Mitsuru Takami: data collection/statics. Yusuke Sonoda: data collection/statics. Hiroyuki Takahara: data collection/statics. Kazutaka Nakasone: data collection/statics. Kyoko Yamamoto: data collection/statics. Yuya Suzuki: data collection/statics. Ken‐ichi Tani: data collection/statics. Hidehiro Iwai: data collection/statics. Yusuke Nakanishi: data collection/statics. Mitsuhiko Shoda: data collection/statics. Atsushi Murakami: data collection/statics. Shogo Yonehara: data collection/statics. Ken‐ichi Hirata: Supervision.This research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.The Section of Arrhythmia is supported by an endowment from Abbott JAPAN, Medtronic JAPAN, and Boston Scientific JAPAN. Ken‐ichi Hirata chairs the Section, and Koji Fukuzawa and Mitsuru Takami belong to the Section. However, the authors have no competing interests to declare that are relevant to the content of this article.


The effluents discharged from Mansoura Company for Resins and Chemicals Industry were evaluated for drinking and irrigation purposes. Calcium-alginate beads were used for effluents treatment in this study. Young male rats were also allowed to drink effluents at different concentrations  and treated 100% effluents with calcium-alginate for 11 weeks. Results indicated high concentrations of some physicochemical parameters and Cd, Co, Fe, Mn, Ni, Pb, and Zn in effluents that exceeded the permissible limits for drinking and irrigation purposes. Treatment by calcium-alginate alleviate heavy metals concentration but did not affect the physicochemical parameters. Depending on effluents concentration, the liver of young male rats showed high accumulation of Fe, Mn, Zn, Pb, Cd, Co, Cu, Cr, and Ni compared to the control group. Serum levels of liver enzymes, total bilirubin significantly increased while total protein, and albumin contents decreased in effluent groups. Liver concentrations of malondialdehyde and protein carbonyl significantly elevated along with significant decrease in superoxide dismutase, catalase, glutathione-S-transferase activities, and glutathione content. Moreover, growth and thyroid hormones were significantly reduced along with significant elevation in thyroid stimulating hormone. This was accompanied by significant decrease in the body weight, especially with 100% effluents concentration compared to control group. Also, histological investigations of both liver and thyroid gland using hematoxylin and eosin showed distortion in the structure of both organs especially with 50% and 100% effluent groups. However, treatment of effluents by calcium-alginate improved these changes. The study revealed that calcium-alginate are effective biosorbents for heavy metals and consequently decrease animal and human health hazards, but further studies are needed to alleviate physicochemical characteristics. The scarcity of freshwater is a major challenge in Egypt that goes with the increase in population growth  is a pioneer in the production of formaldehyde, urea, phenolic resins including molding powder, brake lining, foundry resins, and other chemicals in Egypt, the Middle East, and Africa . This polymer is extracted by treating seaweeds with sodium carbonate solution, where alginic acid is precipitated and sodium salt is formed. In addition, alginate products are known to have high sorption uptake, abundance, high selectivity, and no toxic effects . This factory is located at Sandoub-Elsinbellaween road in Mansoura city , 40 km from Damietta port . The nine samples were taken in clean polyethylene bottles of 10 L capacity, where each bottle was rinsed three times before collecting the sample. Approximately 5 ml of concentrated nitric acid was added to the whole sample to prevent heavy metals from precipitating.The hydrogen ion concentration (pH) value of the samples was determined by an electrical-pH meter in the field . Electrical conductivity (EC) was detected immediately in water samples in the field using a Model C535 multiparameter analyzer.3−) and sulfates (SO42−) were detected according to  were determined using the Meneer method as described in the American Public Health Association  and potassium (K+) were detected by using a flame photometer (Model PH 80B Biologie Spectrophotometer). Calcium (Ca++) and magnesium (Mg++) were estimated using versenate (EDTA) titration method according to  which were measured directly using CORNING (Cole –Parmer model Check 90). Bicarbonate  detected from the equation:SAR =Kelley’s ratio (KR) which defines the harmful impacts of sodium on irrigation water quality was expressed as:KR = Kelley, .Sodium percentage (Na%) was calculated from:Na% =  Wilcox, .The permeability index (PI) which defines water suitability for the irrigation, index is calculated from:PI =  Doneen, .The magnesium adsorption ratio (MAR) was calculated as:MAR = The MRI effluent samples were analyzed for different heavy metals concentration, including cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), lead (Pb), and zinc (Zn). Effluent samples were filtered through Whatman No.1 filter paper before analysis. Bi-distilled deionized water was properly used for washing glassware throughout the study. The metals concentration was detected on a Buck Scientific Accusys 211 series “Atomic Absorption Spectrophotometer”, USA, by an air/acetylene flame system according to standard methods provided by APHA  at wavel2 solution under gentle stirring at 25 °C. Ca-alginate beads were formed upon contact with the Cross-Linker solution. Beads were left overnight to stabilize, then filtered, washed three times with bi-distilled water, and left to dry to be ready for use in effluents treatment.According to EL-Tayieb et al. , sodium Sorption was performed by adding 100 g dried Ca-alginate beads to a volume of 1000 ml effluents sample in a reactor. Sorption was evolved with the mixture under orbital shaking at 25 °C. After 5 h, volume samples were collected from the reactor and metal concentrations were detected again by atomic absorption spectroscopy, USA by an air/acetylene flame system.A total of 50 young male Wistar rats (45 ± 5 g) were obtained from the Egyptian Institute for Serological and Vaccine Production, Helwan, Egypt. The rats were allowed to acclimatize for one week to standard conditions of illumination with a 12 h light and dark cycle at 23 ± 3 °C room temperature and 40 ± 5% humidity. Water and food were consumed ad libitum according to the Ethics Committee of Mansoura University, Egypt. The food consisted of corn, soybean pulp, sunflower seed meal, alfalfa pellets, molasses, vitamins, and minerals that are commonly used for poultry farms.After the acclimation period, rats were randomly divided into five groups (10 rats/each) as follows:Group 1 was the control (CN) group, which received tap water.Group 2 received 10% concentration of MRI effluents (10%.EF).Group 3 received 50% concentration of MRI effluents (50%.EF).Group 4 received 100% concentration of MRI effluents (100%.EF).Group 5 received 100% effluents after treatment with Ca-alginate beads (100%.EF + AG) group.Each group received specific drinking water daily for 11 weeks.The weight of each rat in each group was detected weakly using a digital balance. However, the body weight gain was calculated from the equation:Body weight gain = Final body weight - initial body weight.At the end of the experimental period, rats were weighed and sacrificed. From each rat, two blood samples were collected, and the first was received in an EDTA tube to assess hematological indices. However, the second sample was collected in a sterilized tube without anticoagulant and centrifuged at 860 × g for 15 min. Nonhemolyzed sera were separated and kept at − 20 °C until analysis.Blood samples collected on EDTA were used to determine red blood cells (RBCs) count, hemoglobin (Hb) content, hematocrit (Hct%), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBCs) count, and platelets (PLts) count using a Sysmex Cell Counter , according to Dacie and Lewis .Serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) activities were estimated using the enclosed method in Spinreact kits . However, serum total bilirubin (TB) level and albumin (Alb) content were detected by using the method of Diamond Diagnostics, Co. Kit, Cairo, Egypt. Serum total protein (TP) content was determined using a kit purchased from Bio-Diagnostic Co. Dokki, Giza, Egypt.® 2000 systems. An ELISA kit was also used to detect serum thyroid stimulating hormone (TSH) according to the manufacturer’s protocol of LDN, Germany.Enzyme-linked immunosorbent assay (ELISA) kits were used to detect serum growth hormone (GH) concentrations in different investigated groups, according to the manufacturer’s protocol of MyBioSource, USA. The levels of serum total triiodothyronine (T3) and thyroxine (T4) were measured according to the method of chemiluminescent enzyme immunoassay enclosed in Siemens Healthcare Diagnostics products LTD- IMMULITEImmediately after collecting blood samples, rats of each group were dissected, the thyroid gland and a part of the right lobe of the liver were separated, for histological studies. However, approximately 0.5 g from the left lobe of liver was taken, homogenized in 5 ml cooled distilled water, and centrifuged at 860 × g for 15 min. The clear supernatant was collected and stored at − 20 °C until analysis of antioxidants and oxidative stress markers. A third sample of the liver tissue was kept at − 20 °C for heavy metal analysis.The liver superoxide dismutase (SOD) and catalase (CAT) activities, in addition to reduced glutathione (GSH) content and malondialdehyde (MDA) concentration, were estimated using the specific enclosed methods of Biodiagnostic Co. Dokki, Giza, Egypt. However, an ELISA kit was used to determine glutathione S-transferase (GST) concentration in the liver according to the manufacturer’s protocol of Cusabio, USA. Moreover, the protein carbonyl (PC) level in the liver was assessed according to the manufacturer’s protocol enclosed in the Cayman Chemical Kit .3 was added. The conical flask was allowed to heat on a hot plate at 95 °C until white fumes evolved and then left to cool at room temperature. After cooling, 3 ml of concentrated H2SO4 was added and then heated until the release of orange fumes. The flask was allowed to cool again at room temperature, and then 2 ml of HClO4 was added and heated until the solution was reduced to approximately 5 ml. Each digested sample was filtered through 0.45-µ Millipore membrane filter paper diluted to 50 ml with deionized water in a volumetric flask and analyzed using a Buck Scientific atomic absorption spectrometer  equipped with an air/acetylene flame and hollow cathode lamps for the analyzed elements.Nitric-sulfuric-perchloric acid digestion is an approach that is partly modified from that of Tinggi et al. . One graEvaluation of the structural changes in the liver and thyroid gland via light microscopy was achieved by hematoxylin and eosin (H&E) staining. According to the method of Kiernan et al. , the tisp < 0.05.The data were analyzed using the GraphPad Prism software program  using one-way ANOVA followed by Tukey test. All the results were recorded as the mean ± SD. The Pearson correlation coefficient was used to estimate the correlations between physicochemical characteristics and/or the selected heavy metals in untreated and treated effluent samples. Statistically significant data were considered at The appropriateness of effluents for drinking and irrigation purposes was evaluated through physicochemical characteristics. A statistical summary of the physicochemical parameters of the effluent samples is listed in Table The maximum and minimum electrical conductivity (EC) values were 3.56 and 2.06 dS/m for untreated MRI effluents (EF) and 3.87 and 3.17 dS/m for effluents treated with Ca-alginate (EF + AG), respectively. Moreover, by comparing the mean EC values of different effluent samples to WHO limits for drinking water and FAO limits for irrigation water, the mean EC values (2.802 ± 0.59 and 3.600 ± 0.30) dS/m of both untreated (EF) and treated effluents (EF + AG), respectively exceeded the WHO limits. However, EF samples were within FAO limits for irrigation water, but EF + AG samples were greater than the FAO limits.The pH values of untreated MRI effluent samples, as well as treated effluents, exhibited mostly acidic features. The maximum and minimum recorded pH values were  and , respectively. The mean pH value for the untreated samples (5.71 ± 1.77) was found to be below the permissible limits recommended by the WHO for drinking water and FAO for irrigating water. However, that of treated effluent samples (6.46 ± 1.59) was within these limits.Total dissolved solids (TDS) values showed maximum and minimum values . The average of both samples (1921 ± 618.3 and 1992 ± 518.1) mg/L exceeded the permissible drinking water limit recommended by the WHO but was within the limits of the FAO for irrigation water.3− contents of the effluent samples were 162.5 and 94.79 mg/L and 189.59 and 67.1 mg/L for EF and EF + AG, respectively. However, the maximum and minimum values of the Cl− content of the samples were 817.6 and 483.9 mg/L and 984.4 and 631.0 mg/L for EF and EF + AG, respectively. The SO4−− content of the investigated water samples varied between 621.6 and 472.1 mg/L for EF and 519.7 and 326.1 mg/L for EF + AG. The mean values of HCO3− in EF and EF + AG  were within the permissible limits of WHO for drinking water and FAO for irrigation water. However, the mean values of both Cl− and SO4−− in EF and EF + AG  exceeded WHO drinking water limits in addition to FAO limits for SO4−− values.The maximum and minimum HCO+ concentrations were 657.2 and 434.65 mg/L for EF samples and 830.49 and 418.77 mg/L for EF + AG, respectively. However, K+ concentrations showed maximum and minimum values  and  mg/L for EF and EF + AG, respectively. Moreover, the maximum and minimum Ca++ concentrations of the effluent samples were  and  mg/L for EF and EF + AG, respectively. The Mg++ concentrations of the effluent samples ranged between  and  mg/L for EF and EF + AG, respectively. Furthermore, for both untreated and treated effluents, the mean Na+ concentrations (551.0 ± 104.8 and 681.1 ± 185.2) respectively increased above the WHO drinking water limits but were within the FAO irrigation limits. The mean concentrations of K+ ions in both samples (6.63 ± 1.7 and 6.90 ± 0.96) were within WHO and exceeded FAO limits. However, the mean values of Ca++  and Mg++  in EF and EF + AG samples, respectively, were within both WHO drinking water and FAO irrigation water limits.The maximum and minimum NaThe chemical oxygen demand (COD) values of the collected effluent samples showed maximum and minimum values between  for EF and  mg/L for EF + AG. Moreover, the COD mean values (889.5 ± 37.37 and 878.5 ± 41.62) for EF and EF + AG, respectively, exceeded the permissible drinking water levels of the WHO. However, COD limits for irrigation water were not included in FAO standards.It should also be mentioned that the treatment of effluents with Ca-alginate did not statistically show any significant differences in the estimated physicochemical parameters compared to untreated effluent samples.Table Table −/EC, Ca++/EC, Ca++/TDS, and Ca++/Cl−. However, the estimated heavy metals in effluent samples exhibited positive and negative but nonsignificant correlations with the detected physicochemical parameters. This could be seen for example, in pairs of Cr/pH (r = 0.229), Fe/Cl− (r = 0.030), Mn/ SO4−− (r = 0.186), Co/Cl−(r =  − 0.364), Pb/Na+(r =  − 0.149), Cd/Mg++ (r =  − 0.082), and Ni/COD (r =  − 0.203). On the other hand, the elemental pairs showed significant positive correlation between all investigated metals.The Pearson correlation coefficient matrix (r) was used to determine the interaction between physicochemical characteristics and heavy metal concentrations, as well as each other in untreated and treated MRI effluent samples Table . The phyThe obtained data in Table The present study showed significant changes in the investigated parameters along all MRI effluent concentrations. ALT, AST, and TB increased significantly especially at 50%EF and 100%EF groups compared to the control group. However, TP and Alb decreased significantly with all the studied concentrations of MRI effluents. Treatment of 100%EF samples with alginate (100%EF + AG) showed significant improvement in liver function parameters represented in significant decrease in ALT, AST, and TB along with an obvious increase in TP and Alb contents compared to all effluent concentration groups as shown in Fig. .Fig. 2LiThe obtained data showed obviously decreased liver antioxidant activities of CAT, SOD, GST, and GSH content with increasing concentrations of MRI effluents compared to the control group. This decrease was mostly significant with 50%.EF and 100%.EF groups. On the other hand, oxidative stress markers (MDA and PC) showed significant increase with drinking different concentrations of MRI effluent groups compared to control. Treatment with Ca-alginate exhibited a significant increase in the studied antioxidants in the liver of 100%.EF + AG rat group which also exhibited a significant decrease in MDA and PC compared to the 50%EF and 100%.EF groups Fig. .Fig. 3LiTo follow the rate of growth experimentally, Table The present results illustrated in Table The investigated hematological parameters  decreased significantly with increasing concentrations of MRI effluents in drinking water, especially at 50% and 100% concentrations. However, the pLts count increased significantly at the same concentrations. On the other hand, 100%EF + AG group showed significant improvement in hematological indices compared to the untreated effluent group, especially in RBCs, Hb, Hct, MCV, and MCHC Table .Table 8HIt should be mentioned that the obtained results showed nonsignificant difference between the treated EF group (100%.EF + AG) and the control one in most investigated parameters, except GST activity, GH levels, MCH, MCHC, and WBCs count.Histopathological investigations in the livers of different groups showed normal liver architecture with distinct hepatocytes consisting of hepatic stands and blood sinusoids surrounding intact central veins Fig. a. The raThe normal structure of the thyroid gland was represented in the control group, where parenchymal cells appeared in two types: follicular and parafollicular cells. Follicular cells (thyroid follicles) of various sizes lining a central colloid-filled lumen-stained pink Fig. a were obThe direct discharge of untreated industrial effluents into drains, canals, or rivers worsens the problem of water pollution. The suitability of water used from drains, canals for irrigation purposes, and watering livestock was taken into consideration in this study. Estimation of certain environmental factors, such as pH, EC, TDS, and anionic and cationic components of water, plays an important role in water evaluation  and cations (Na+ and K+) components in MRI effluent samples were higher than the WHO and FAO limits for drinking and irrigation water. Tariq et al.  WHO, . The EC q et al.  reflecteq et al.  mentione+, Ca++, Mg++, and HCO3− in MRI effluents in this study were used to determine the quality of effluents for irrigation through detecting SAR, KR, Na%, PI, and MAR. Obtained results showed unsuitability of MRI effluents for irrigation purposes due to high values of SAR, KR, Na%, and PI. These results agreed with Veena and Satheeshkuamar  compared to the control group. This may be due to the absorbance of metals by intestinal cells that pass through the blood to the liver for the detoxification process by metallothionein protein synthesized in the liver  , especially at 100%.EF group compared to control. David et al.  attributBioremediation using algae has been successfully used as a biosorbent for heavy metal detoxification  above the WHO and FAO limits, indicating the unsuitability of MRI untreated effluents for drinking or irrigation purposes. Treatment of effluents by using Ca-alginate beads for 5 h represented an effective and safe biosorbent for heavy metal decontamination and purification of effluents. However, non-significant changes in the physicochemical characteristics of effluents were observed with treatment. Moreover, in a process mimicking livestock exposure to polluted effluents at different sites, young rats exposed to different effluent concentrations showed significant increase in heavy metals concentration accumulated in liver of investigated groups. This was accompanied by significant disturbance in liver function and oxidative system along with disruptors in growth and thyroid hormones leading to growth retardation and body weight loss. All these foundations were supported by histopathological investigations in the liver and thyroid gland. This may indicate that closer villages to the MRI company, greater risks will be found, whether in terms of usage in irrigation or for watering livestock. However, rats received treated effluents with Ca-alginate beads showed significant amelioration in different investigated parameters compared to effluent groups. Nevertheless, Ca-alginate beads should be applied to MRI effluents for heavy metals biosorption, while further studies are needed to improve the physicochemical characteristics of effluents.


In recent years, researchers have become increasingly interested in developing natural feed additives that can stabilize ruminal pH and thus prevent or eliminate the risk of severe subacute rumen acidosis. Herein, 3 experiments were conducted using a semi-automated in vitro gas production technique. In the experiment (Exp.) 1, the efficacy of 9 plant extracts (1.5 mg/ml), compared to monensin , to counteract ruminal acidosis stimulated by adding glucose (0.1 g/ml) as a fermentable carbohydrate without buffer was assessed for 6 h. In Exp. 2, cinnamon extract (CIN) and MON were evaluated to combat glucose-induced acidosis with buffer use for 24 h. In Exp. 3, the effect of CIN and MON on preventing acidosis when corn or barley grains were used as substrate was examined.P < 0.05) pH compared to control (CON). Both CIN and MON significantly increased the pH (P < 0.001) but reduced cumulated gas production (P < 0.01) compared to the other treatments. In Exp. 2, the addition of CIN extract increased (P < 0.01) pH value compared to CON at the first 6 h of incubation. However, no significant differences in pH values between CIN and CON at 24 h of incubation were observed. The addition of CIN extract and MON decreased (P < 0.001) lactic acid concentration and TVFA compared to CON at 24 h. The CIN significantly (P < 0.01) increased acetate: propionate ratio while MON reduced it. In Exp. 3, both CIN and MON significantly increased (P < 0.05) ruminal pH at 6 and 24 h and reduced lactic acid concentration at 24 h compared to CON with corn as substrate. However, CIN had no effect on pH with barley substrate at all incubation times.In Exp. 1, cinnamon, grape seeds, orange, pomegranate peels, propolis, and guava extracts significantly increased (It can be concluded that CIN can be used effectively as an alternative antibiotic to MON to control ruminal acidosis when corn is used as a basal diet. Ruminal acidosis is a prevalent digestive disorder in beef and dairy cattle, particularly during transition periods . These aSeveral approaches used to control ruminal acidosis disorder involve using feed additives such as ionophores. Monensin (MON) is the most common ionophore used in ruminant diets that improve ruminal microbial fermentation . Also, iRecently, several researchers have focused on using botanical compounds as potential and safe alternatives for antibiotics in ruminant diets. Botanical extracts are herbal plants with high content of plant secondary compounds (PSCs) such as saponin, essential oils, and phenolic compounds  , 14. TheHowever, the influence of plant extracts and PSCs on rumen acidosis is still scarce, and some studies noticed that lactic production bacteria were inhibited by the use of ethanolic extracts of Australian plants and essential oils , 22. TheWe hypothesized that these plant extracts would control lactic acid production in the rumen by stimulating lactic acid utilizing bacteria and inhibiting lactic acid-producing bacteria, same as MON. Thus, this study aimed to initially screen nine plant extracts against in vitro rumen acidosis. (ii) Then, evaluating the extent of protection against acute and sub-acute acidosis by selecting the promising plant extracts from the initial screening, with effects similar to MON. (iii) Evaluating selected plant extracts on the degradation of different ingredients (barley and corn grains) in vitro.The current research was performed at the Animal Nutrition Laboratory of Animal and Fish Production Department, Faculty of Agriculture (El-Shatby), Alexandria University.Punica granatum), orange peels (Citrus sinensis), and grape seed (Vitis vinifera) were obtained from a food factory in the industrial region of Borg EL-Arab, Alexandria. Tree leaves of olive (Olea europaea), guava (Psidium guajava), and mango (Mangifera indica) were collected from a private orchard in Alexandria, and green tea leaves (Camellia Sinensis) from Abosheba Company, Jeddah, Saudi Arabia. Propolis powder was supplied by a trading Company Henan, China, and the grounded cinnamon (Cinnamonum cassia) was purchased from Royal Spices Company .Nine plant extracts were used in the present study: Agricultural by-products of pomegranate peels  at a temperature of 50 °C as described by Zarina and Tan (2013). Extracts were filtered by Whitman filter paper No, 42 125 mm) and evaporated under pressure at 40 °C using rotary evaporation . Extracts were freeze-dried and stored at 4 °C for later assay. The contents of plant extracts of total phenols (TPs), total tannins (TTs), condensed tannin (CT), and total flavonoids (TFs) were determined calorimetrically  in each experiment at an abattoir belonging to the Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria, Egypt. Collection of ruminal content from slaughtered animals saves money and overcomes the need for surgical cannula in live animals with complete adherence to animal protection law . Also, t, orange peels extract, grape seed extract, olive leaf extract, guava leaf extract, mango leaf extract, green tea leaf extract, propolis extract, and cinnamon extract (CIN). The tested concentration of the plant extracts has been selected based on a preliminary experiment (data not shown). In the earlier experiment, we have tested the 9 plant extract at a concentration of 1 mg/ml following the study of Durmic, et al. [Nine plant extracts compared to MON were evaluated against ruminal acidosis using the procedure described by Hutton, et al. . A semi-, et al. . However, et al. . Plant e2 and sealed with a rubber stopper and aluminum crimps. The pressure in the bottle’s headspace was adjusted to zero by inserting a 23 G needle in the stopper of the bottles, then all bottles were transferred to the incubator and incubated at 39 °C for 6 h.A total of 45 mL of the ruminal inoculum was placed in the 120-mL serum bottles and then dissolved plant extracts were added at 450 μl per 45 ml of rumen fluid. The initial pH of mixed rumen fluid was measured. Dimethyl sulfoxide was added at 450 μl of solvent to the control bottles. All bottles were gassed with COThe incubated bottles were removed from the incubator after 2, 4, and 6 h to measure gas pressure by inserting a 23 G needle in the headspace using a pressure transducer and data logger  according to Mauricio, et al. . The bot’s buffer [This experiment assessed the extended protection against sub-acute and acute acidosis for CIN compared to MON by measuring VFA and lactic acid production. The incubation conditions of the second experiment were similar to the first experiment with the following differences: fermentation time was increased from 6 to 24 h. Buffer was used in the assay because the pH value may decline due to the accumulation of organic acids over 24 h. The incubation media (45 ml) was bottled consisting of 22.5 ml of ruminal fluid and 22.5 ml of McDougalls buffer . The gluCumulative gas production was measured after 2, 4, 6, 9, 12, and 24 h of incubation. Carefully, 1 ml of liquid phase was taken by inserting 23 G needle in rubber stopper of the bottle after 6, 12, and 24 h of incubation to estimate pH value. At the end of incubation time (24 h), 1 ml of liquid phase was transferred into 1.5 ml Eppendorf tube, then 200 μL of meta-phosphoric acid 25% (w/v) was added and stored at − 20 °C for later determination of VFA concentration according to the methods described by Palmquist and Conrad . SamplesThe effect of CIN and MON supplementation on in vitro fermentation of barley and corn grains was investigated. Barley and corn grains were grounded to pass a 1 mm screen and used as substrates. About 4.5 g of ground grains in triplicates were placed into serum bottles to induce rumen acidosis, according to Dennis, et al. . The incij = μ + Ti + eij where: μ is the overall mean, Ti is the treatment, eij is the random error term. Data of EXP. 2 were statistically analyzed by factorial arrangement using the following Model 1: Yijk = μ + Ei + Tj + ETij + eijk, where Yijk = the measured parameter, μ = the overall mean, Ei = the main effects of treatment  ith treatment, Tj = the main effect of jth incubation time, ETij = the interaction between the ith treatment and incubation time and eijkl = random error. Data of EXP. 3 were analyzed as in EXP. 2 to identify the main effects of treatment, substrate (corn and barley), and incubation time were included in the model with the correct interactions. The differences between treatments were considered significant at P < 0.05. The Tukey’s multiple range post hoc test was used for pairwise comparisons.The data was statistically analyzed using the MIXED procedure of SAS . In EXP 1, the following model was assumed: YP < 0.05) increased the pH values except mango, green tea, and olive extracts. The maximum increment in pH was recorded in CIN and MON. The effect of other plant extracts on pH was as follows: pomegranate peels and grape seeds extracts > guava and orange peel extracts > propolis extract.As presented in Fig. P < 0.01) was observed with MON (97.69 kPa) and cinnamon (102.02 kPa) compared to control (120.98 kPa). On the other hand, no significant differences were observed with other plant extracts on cumulative gas production compared to control.As shown in Fig. P < 0.001) was significant with ruminal pH. The addition of CIN significantly increased (P < 0.01) ruminal pH (6.14) compared to CON (5.88) at the first 6 h of incubation. However, there were no significant differences in pH values between CIN and CON at 12 and 24 h of incubation. On the other hand, ruminal pH was higher (P < 0.01) with MON than CON and CIN in all incubation times  effect for treatments, time, and their interaction on cumulative gas production (Table P < 0.01) reduced cumulative gas production. On the contrary, MON significantly decreased (P < 0.01) cumulative gas production in all incubation times compared to CON and CIN.Also, there was a significant (on Table . No signP < 0.01) in MON (45.60 mM) than CIN (70.64 mM) or CON (86.80 mM). Both CIN and MON decreased (P < 0.01) total VFA concentration (83.89 mM and 65.73 mM) compared to control (100.25 mM) after 24 h of incubation. The addition of CIN increased the molar concentration of acetate and reduced the molar concentration of propionate (P < 0.01) compared to CON and MON. Consequently, the highest value of acetate: propionate ratio (4.17) was found with CIN compared to CON (3.75) and MON (3.46).As demonstrated in Table . No significant (P > 0.05) differences were observed in the effect of treatment and substrate on cumulative gas production. No interactions were observed between treatment and substrate. On the contrary, a significant (P < 0.001) interaction between treatment × time and substrate × time was observed.Effects of MON and CIN addition on cumulative gas production (kPa) using corn or barley as substrate are shown in Fig. 3P < 0.001) between substrate and time on pH value was observed. There was a significant effect of substrate (P < 0.001) on ruminal pH where barley reduced overall ruminal pH (4.64) compared to corn (4.98). Also, an interaction (P < 0.01) was observed between treatment × substrate × time. When corn was used as a substrate, the addition of CIN and MON significantly increased (P < 0.05) ruminal pH compared to control at 6 h and 24 h of incubation; however, no effect of MON and CIN on pH values at 12 h compared to CON. On the other hand, when barely used as a substrate, MON did not affect pH at 6 and 12 h; however, it increased (P < 0.01) ruminal pH at 24 h compared to CIN and CON.Changes in ruminal pH values with corn and barley as substrates at different incubation times are presented in Table P < 0.01) interaction was found between substrate and treatment, resulting in reduced lactic acid concentration for CIN treatment compared to CON and MON when corn was used as a substrate. The effect of the substrate was significant (P < 0.01) as barley caused a higher lactic acid and acetate production compared to corn. However, no effect of the substrate was found on propionic acid and total VFA. MON increased (P < 0.01) propionic acid and reduced acetate concentrations compared to CON and CIN with both substrates (corn and barley), resulting in lower acetate: propionate ratio for MON compared to CON and CIN with both substrates. Also, MON significantly reduced (P < 0.01) butyrate concentration compared to CIN and CON with both corn and barley as substrates.The effects of MON and CIN on rumen fermentation products at 24 h incubation time using corn and barley as substrates are presented in Table streptococcus bovis) [streptococcus bovis prevented pH decline and stimulated lactic acid fermenting bacteria like Megasphera elsdenii [Nine plant extracts were used to examine their efficiency in preventing acidosis conditions. In Exp.1, an in-vitro bioassay  was useds bovis) . The inhelsdenii .The plant extracts used in this screening bioassay were chosen for their higher content of phenolic compounds. Xia, et al.  and SaleCinnamomum and belonging to Lauraceae family [(Onobrychris viciifolia) decreased the growth of several ruminal bacteria such as Butyrivibrio fibrisolvens, Streptococcus bovis, Prevotella ruminicola, and Ruminobacter amylophilis. Such inhibitory effects are due to the binding of condensed tannins to the bacterial cell wall and deactivating the cell-bound extracellular enzymes [In this study, CIN efficiently increased pH more than the other plant extracts. Cinnamon is derived from a Greek word that means sweet wood obtained from the inner bark of trees genus e family . The effe family . Therefoe family , 49. Jone family  reported enzymes . Besides enzymes . Cinnama enzymes .In EXP.1, CIN achieved the most favorable effect on ruminal acidosis. Hence, it was chosen to be evaluated either with glucose (EXP.2) or grain substrates (EXP.3). In EXP. 2, CIN maintained greater pH than control due to its high content of antimicrobial compounds against lactic acid-producing bacteria. However, this action could not be sustained up to 12 and 24 h of incubation. When glucose was used as a substrate (EXP. 2), the MON addition increased the pH and decreased lactic acid concentrations compared to CON up to 24 h of incubation. But, MON could not raise the pH above 5.0, indicating that the protection assay was truly effective against sub-acute acidosis up to 6 h for CIN and 12 h for MON. Furthermore, a similar trend was obtained when glucose was replaced by corn or barely (EXP.3). This could be attributed to the higher concentration of substrates  and grains  that were used (4.5 g/45 ml) to induce acidosis.Under normal fermentation conditions, lactic acid is an intermediate product in the rumen in carbohydrate fermentation that is readily converted into VFA by lactate-consuming bacteria . HoweverMegasphera elsdenii that convert lactic acid to propionate, thus lowering the acetate to propionate ratio [The total VFA concentration was not affected by CIN when corn or barely was used as substrate (EXP. 3), indicating that CIN maintained balanced microbial activity. However, CIN reduced total VFA concentration when glucose was the substrate (EXP. 2). The inconsistent effect of CIN in EXP. 2 and 3 on total VFA is not understood. This effect can be attributed to the difference in the substrate used and the experimental conditions. The finding that CIN increased the acetate/propionate ratio in EXP. 2 was contrary to MON, which reduced the acetate/propionate ratio. The selective effect of MON against lactate-producing bacteria enhanced the activity of lactate-fermenting bacteria such as te ratio . Cardozote ratio  reportedte ratio , comparete ratio . Also, Cte ratio  reportedAnother explanation for the greater acetate concentration In EXP. 2 is that the tannins present in CIN could stimulate bacteria to produce acetate from glucose. Zhao, et al.  reportedAs expected, barley reduced rumen pH value and increased lactic acid concentration compared to corn, which agrees with Fulton et al. (1979), who reported that steers fed wheat as concentrate had lower ruminal pH than steers fed a corn-based diet. Despite having more starch than barley, corn’s starch is less available for degradation by rumen bacteria due to the less degradable protein membrane surrounding its starch granules . This inCompared to other plant extracts, CIN was the best plant extract after MON to prevent rumen acidosis in-vitro. Also, cinnamon altered ruminal microbial fermentation by increasing the acetate to propionate ratio. This may indicate that CIN is a beneficial additive to transition diets for lactating animals. Cinnamon had beneficial effects when corn was used as a substrate on rumen fermentation profiles by increasing ruminal pH and decreasing lactic acid concentration. Further investigations are required in-vitro and in-vivo to study the impact of CIN on controlling ruminal acidosis.


The potential of the miniJoint in predicting in vivo efficacy of drug treatment is confirmed by testing the “therapeutic effect” of the nonsteroidal anti‐inflammatory drug, naproxen, as well as four other potential disease‐modifying OA drugs. The data demonstrate that the miniJoint recapitulates complex tissue interactions, thus providing a robust organ chip model for the study of joint pathology and the development of novel therapeutic interventions.Diseases of the knee joint such as osteoarthritis (OA) affect all joint elements. An in vitro human cell‐derived microphysiological system capable of simulating intraarticular tissue crosstalk is desirable for studying etiologies/pathogenesis of joint diseases and testing potential therapeutics. Herein, a human mesenchymal stem cell‐derived miniature joint system (miniJoint) is generated, in which engineered osteochondral complex, synovial‐like fibrous tissue, and adipose tissue are integrated into a microfluidics‐enabled bioreactor. This novel design facilitates different tissues communicating while still maintaining their respective phenotypes. The miniJoint exhibits physiologically relevant changes when exposed to interleukin‐1 A miniature synovial joint‐mimicking system (miniJoint) is established in a microfluidics‐enabled bioreactor, in which human mesenchymal stem cell‐derived bone‐cartilage complex, synovial‐like fibrous tissue, and adipose tissue are integrated and show active crosstalk. The data demonstrate the potential of miniJoint as a new model for the study of joint pathology and the development of novel therapeutic interventions. The articular cartilage is primarily anatomically bound to the subchondral bone, forming an osteochondral unit. The biophysical and biochemical communication between cartilage and bone plays a crucial role in maintaining joint homeostasis and mediating pathological changes. Cartilage, synovium, and other intraarticular tissues are bathed in synovial fluid, the lubricant found in the joint cavity, which serves as the vehicle for mediating the molecular and cellular crosstalk among these tissues. While conventional in vitro tissue culture platforms and different animal models have significantly increased our understanding of disease etiologies and pathogenesis, some critical challenges remain. For example, multicomponent, real‐time, and bi‐directional crosstalk information has rarely been observed in current in vitro models of the joint diseases. Moreover, there are significant differences in pathology and drug responses between animal models and humans. Therefore, a human cell‐derived model of the joint is urgently needed to enhance our understanding of the pathogenesis of joint diseases in humans and facilitate the discovery of novel drug treatments.Diseases of the knee joint such as osteoarthritis (OA) are highly prevalent, debilitating disorders that severely compromise the quality of life, creating a significant socioeconomic burden for affected individuals.1 Alth Models generated using this technology aim to emulate the in vivo microenvironment characterized by diverse cellular composition, tissue‐specific extracellular matrix, and interactive biochemical and physical signals. Using human cells and integrating multiple tissues, the limitations of current animal models and in vitro culture assays can be partially overcome. As the first step toward whole joint construction in vitro, we have previously created a biphasic osteochondral model, which demonstrates active crosstalk between bone and cartilage throughout tissue maturation and disease progression. Recently, a chondrocyte‐derived cartilage chip was developed by Occhetta et al. to simulate the influence of mechanical load on cartilage.Tissue‐on‐a‐chip technology offers a 3D engineered system that mimics the physiological microenvironment.6 Mode Here, we report the development and characterization of a 3D, multicomponent, and human cell‐based miniature synovial joint system (miniJoint)  separately and then integrated into a custom‐designed bioreactor to generate the complete miniJoint , a classic cytokine widely used to induce OA‐like features in vitro, was introduced to simulate the inflammatory features observed in most joint diseases  can be isolated from different human tissue sources and expanded to a relatively high number without significantly losing their original stem cell capabilities, they are an ideal cell source for creating the miniJoint. Additionally, hMSCs have demonstrated robust osteogenic, chondrogenic, and adipogenic differentiation capacity.11 In t Figure . The abis Figure . Transcr22.1 The scaffold also acted as a barrier to minimize free medium exchange between the top and bottom flows  supplementation and varying dexamethasone and bone morphogenetic protein 7 (BMP7) treatment times. Based on the results shown in Figure The biphasic nature of native OC tissue, consisting of osseous and chondral components in direct contact, presented technical challenges, versus a uniform construct incorporating a single cell type. Previously, we had successfully generated this heterogeneous tissue by employing a dual‐flow OC bioreactor, in which osteogenic and chondrogenic media were perfused separately through the top and bottom streams.8a Oure Figure . To furtβ3) in the chondrogenic medium (CM) and BMP7 in the osteogenic medium (OM), respectively. Quantitative modeling of growth factor diffusion in the GelMA scaffold showed that the concentrations of TGFβ3 and BMP7 decreased by 98.95% and 100%, respectively, over a distance of 4.8 mm . From one end of the scaffold to the other, the corresponding dye concentration, indicated by fluorescence intensity, was found to decrease by 99.84% and 99.94%, respectively  expression levels, and the chondral phase (OC‐C) showed much higher collagen type II (COL2) and aggrecan (ACAN) expression  had significantly higher osteocalcin , tenascin C (TNC) and versican (VCAN)  Figure . In addi2.2β3 was required to maintain the cartilaginous phenotype of the engineered cartilage tissue component . As shown in Figure −1 IL‐1β treated fibroblasts displayed an inflammatory phenotype. Also, the conditioned medium collected from IL‐1β treated fibroblasts was able to induce the degeneration of engineered cartilage  in disease pathologies by testing whether and how other surrounding tissues would be altered. Abundant evidence indicates that inflammation of the synovium (synovitis) is critical in the pathogenesis of joint diseases.12 We β, we next created “synovitis” in the miniJoint and examined the influence of inflamed SFT on other tissues. As shown in Figure β was introduced to the FM stream in the miniJoint to only stimulate SFT, while other tissues were not directly exposed to IL‐1β treatment. After 7 days, the viability of cells in SFT was evaluated by LIVE/DEAD and alamarBlue assays  in AD. Interestingly, enhanced expression of MMP‐13 was concomitant with the upregulation of expression of osteopontin (OPN) and ALP in OC‐O in the inflamed miniJoint, suggesting a possible remodeling process. The tissues in the inflamed miniJoint showed consistently upregulated expression of nerve growth factor (NGF), a key mediator of the sensation of joint pain.After validating the function of fibroblasts in responding to IL‐13 Figure . RNA‐Seqin Table 13 Upr3 Figure , signifyβ treated SFT, indicating an increase in the number of apoptotic cells  assay revealed a considerably higher level of DNA fragmentation in IL‐1s Figure . The IL‐FigureIn the design of the miniJoint, the crosstalk between SFT and other tissues was mediated primarily through the SM; we therefore measured the levels of selected biomarkers in SM using Luminex immunoassay and ELISA (for targets not available in Luminex) Figure5a,b. ThTaken together, these data supported the successful generation of inflammatory and degenerative features of joint diseases in the miniJoint. The results also demonstrated that OC and AT microtissues were responding to the pathological changes of SFT, implying the active crosstalk within miniJoint. As shown in Figure; and Table  We next performed pathway enrichment analysis based on the DEGs in both miniJoint  introduced in the FM stream. Comparative evaluations of OC‐C microtissues harvested from the challenged chips and the healthy (no IL‐1β insult) control were carried out by RNA‐Seq . NPX treatment was found to effectively mitigate the inflammation induced by IL‐1β treated SFT. For example, the expression levels of MMP‐1, 3 and 13 in SFT were found to be downregulated after NPX treatment , a gene that encodes IL‐1RA that can block the binding of IL‐1 to its receptor. NPX treatment also reduced the expression of ADIPQ and LPL in AD, as well as suppressed ALP expression in OC‐O.To validate the utility of the miniJoint chip as a platform for drug screening, it is important to assess the impact of agents with known clinical efficacy. Since there are no DMOADs approved by regulatory bodies, naproxen (NPX), a nonsteroidal anti‐inflammatory drug (NSAID) commonly prescribed to OA patients, was selected for the study. NPX was administered into all streams of the inflammatory miniJoint at 5 × 10” Figure . RNA‐Seqt Figure , which wt Figure , indicatt Figure . NPX treFurthermore, analysis of the SM revealed that, upon NPX treatment, concentrations of MMP‐1, MMP‐2, MMP‐3, MMP‐13, IL‐6, IL‐13, PGE‐2, CTX‐II, POSTN, CXCL‐1, and VEGFR2 were lower, compared to the control inflamed miniJoint Figure . In addi2.5−1), SM04690 , sclerostin , and IL‐1RA (250 ng mL−1). To simulate the primarily intra‐articular application of these drugs in clinical trials, they were added only to the SM . It was found that all the drugs tested resulted in statistically significant upregulation of chondrogenic genes, including COL2 and ACAN. In addition, these drugs also induced significant downregulation of gene expression of the inflammatory marker IL‐6 as well as the degenerative marker a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), a gene that encodes aggrecan‐degrading enzyme . Therefore, when cultured in the miniJoint chip (day 28–56), the OC‐C was maintained in a medium with a low level of TGF (0.5 ng mL−1), which significantly decreased the level of hypertrophy in OC‐C, as supported by the immunostaining results in Figure  Following the osteochondral tissue chip's success, the next phase was to incorporate additional joint tissues found within the knee joint. To determine which tissues would be included in our study, two primary joint diseases were considered, OA and rheumatoid arthritis (RA). In RA, synovium inflammation, also called synovitis, is well known as a significant disease contributor. Recently, synovitis has been found to be an active component in OA pathology as well. Therefore, we first elected to include the synovium in addition to the osteochondral tissue. The selection of adipose tissue as the fourth miniJoint element was inspired by the association between obesity and OA, and the contribution of adipose tissue to joint diseases was further shown in several recent animal studies. Adipose tissue produces adipokines as well as major inflammatory cytokines such as IL‐1β and IL‐6. Serum levels of these cytokines are elevated in OA patients, suggesting that metabolic disruption of adipose tissue may play a role in OA severity and progression. The infrapatellar fat pad (IPFP), an anatomically unique adipose tissue located in the knee joint, has been shown to undergo changes during the pathogenesis of OA. However, the exact role of adipose tissue in OA disease progression requires further investigation. While synovium and adipose tissue represent promising additions to our study, other joint tissues, including muscle, meniscus, and intraarticular ligament, also contribute to normal joint function and physiology. These tissues have major biomechanical functions and their injuries caused by abnormal mechanical loading can eventually impact cartilage and bone and lead to joint diseases. These tissues may be included in the future development of joint organ chips.Like the development of other tissue/organ chips,18 eng are also potential cell sources to create the miniJoint in the future.Although primary cells  can be employed to create a joint‐mimicking chip, this approach requires several procedures to collect different tissues to isolate these cells. Also, the relatively low availability and limited expansion potential of primary cells are not compatible with the miniJoint system since it requires many cells. In comparison, MSCs can be extensively expanded and differentiated into four tissue types, which has been shown in the current study. Moreover, another advantage of using MSCs is that the different tissue components are derived from the same starting cell source, whereas the use of primary cell sources or established primary cell lines will almost necessarily involve cells from different individuals, thus presenting potential incompatibilities. Immortal MSC lines, such as human telomerase immortalized cells,25 are Of note, there are no exclusive markers to identify fibroblast‐like synoviocytes. Therefore, we examined several molecules that have been previously shown to be expressed in synovial fibroblasts, including COL1, lubricin, CDH11, β1 integrin, and CD44. In this research, as well as a recent study on synovium‐cartilage chip, macrophage‐like synoviocytes were not included in the SFT; instead, their function was partially realized through IL‐1β treatment. Due to the challenges in 3D culture, especially the requirement of long‐term culturing, macrophages are rarely included in current organ‐on‐a‐chip models. In our previous studies, GelMA‐encapsulated macrophages that were cultured for 1, 7, and 14 days, were subjected to M1 or M2 polarization. We found that macrophage viability and bioactivity decreased with increased culture time. The short life expectancy of macrophages in 3D culture was also observed in another study. To overcome the lack of macrophages in SFT, we used IL‐1β to stimulate the inflammatory component  at the same time, which may mask the effects of potential tissue crosstalk. In addition, as previously described, synovitis is recognized as an important OA and RA feature. In particular, prior synovitis, identified by magnetic resonance imaging, was significantly associated with subsequent OA development, suggesting that synovitis is likely a precursor to radiographic OA. The association between synovitis and further OA progression has also been demonstrated. Therefore, we simulated a “synovitis” model in miniJoint by challenging only SFT with IL‐1β (10 ng/ml), without directly exposing other tissues to exogenous IL‐1β. In concordance with our results in Figure MMP‐13 and IL‐8 was also observed in the mechanical overloading‐induced OA model established in cartilage‐on‐a‐chip by Occhetta et al. Interestingly, we found that the changes in the expression of COL2, ACAN, MMP‐1 and MMP‐13 in OC‐C between the inflamed miniJoint and the healthy control observed here . As shown in Figure  rather, each model recapitulates certain aspect(s) of the pathological changes. The miniJoint developed here can be used to increase the efficiency in selecting drug candidates for large animal studies or clinical trials.While IL‐1elopment,32 sug The Luminex immunoassay employed here allowed us to analyze the known potential biomarkers in a medium‐throughput manner. The SM from the inflammatory miniJoint contained many biomarkers well recognized in the synovial fluid of OA patients, including proinflammatory cytokines, angiogenic factors, and MMPs. In addition, we have found increased CCL2, OPN, CFD, and POSTN levels, which have been newly recognized as OA biomarkers in synovial fluid. The broad array of molecules detected in the SM signifies active involvement of all microtissues, as a result of their crosstalk, in the pathological changes in the inflamed miniJoint. By comparatively analyzing the biomarkers identified by methods such as Luminex and mass spectrometry for miniJoint SM and human synovial fluid, valuable information can be obtained to evaluate the clinical relevance of our miniJoint system. In the current study, we only measured the concentrations of biomarkers in the SM, not the other three streams. The concern was that the use of fetal bovine serum in these media  are being tested in human clinical trials for OA treatment.47 It e Figure . The sma4β simulated inflammation. Furthermore, the potential of miniJoint in assessing and predicting in vivo efficacy of drug treatment was demonstrated by simulating the systemic or intraarticular administration of five drugs that are clinically used or under development for treating joint disorders.We described the engineering of the miniJoint, the first human cell‐derived, multi‐tissue chip with the capacity to mimic both healthy and inflamed knee joints. The miniJoint chip incorporates osteochondral, fibrous, and adipose microtissues, and exhibited physiologically relevant pathological changes when the fibrous tissue experienced IL‐1 Second, in this study we have used the same hydrogel scaffold to derive all microtissues. However, GelMA‐based scaffold with the same stiffness may not be the best option to engineer all miniJoint tissues with desired phenotypes. To further optimize the microtissues, we will explore hydrogels with different mechanical and microstructural properties in the future. The degradability of the scaffold material will also be optimized to facilitate the deposition of cell‐secreted nascent matrix and the replacement of the original, synthetic matrix. Third, although inflammation in the miniJoint was realized or simulated by adding IL‐1β into FM, immune cells such as macrophages have not been included in the miniJoint. As described above, the immune cells can be introduced through including an additional module. Finally, we will create different types of joint disease models in the context of the miniJoint by introducing different stimuli, and test a broad range of drugs to validate the miniJoint's clinical relevance in predicting drug toxicity and efficacy in humans.The clinical relevance of the miniJoint can be further enhanced in the future. First, a mechanically active mechanism may be included in the miniJoint to simulate the physiological weight‐bearing function of the knee joint more effectively. In addition to the direct application of force, mechanical loading can also be partially simulated by directly modulating key mechanotransduction pathways, such as the transient receptor potential cation channel subfamily V member‐4 (TRPV4) pathway, a known mechanosensitive downstream signaling pathway.51 Sec5To design the miniJoint bioreactor 3D models, SolidWorks 2018 software  was used. Each bioreactor part, including the chambers, caps, and inserts, was then additively manufactured by stereolithography using the E‐Shell 450 photopolymer ink and a Vida desktop 3D Printer . The hollow inserts, with a height of 4.8 mm and inner diameter of 3.5 mm, housed the cell‐laden hydrogels, generating modular microtissues Figure . SurfaceDifferent miniJoint tissue components, including OC, AT, and SFT, were generated by differentiating human bone marrow‐derived stem cells (hBMSCs). hBMSCs were harvested from total joint arthroplasty surgical waste with IRB approval (University of Pittsburgh and University of Washington). To minimize donor‐to‐donor difference and obtain sufficient cells, hBMSCs were collected and combined from 20 donors aged 20–87 years old  were resuspended in 15% w/v GelMA at 20 million mL A pulse flow pattern, consisting of 20s fast flow at 12 µL s−1 and 3580 s slow flow at 1/60 µL s−1, was provided by a programmable syringe pump . The OC chip culture was maintained for 4 weeks.To generate OC microtissues, the inserts containing hBMSCs‐laden hydrogel were assembled in a dual‐flow OC chip, and osteogenic medium (OM) and chondrogenic medium (CM) were perfused through the top and bottom streams, respectively   over ≈5 days, trypsinized and plated in T150 flasks at a 2000 cells cm  was first used to generate complementary DNA (cDNA) from the total RNA extracted from the microtissues (RNA extraction described in Supporting Information). The mixture of cDNA samples, forward and reverse primers , ALP , COL2 , and SFT sections in antibodies against lubricin  and CDH11 , overnight at 4 °C. Antibodies against MMP‐13  and IL‐6  were used to examine inflammatory responses by the microtissues. Antigen retrieval was carried out using a 30 min incubation in a combined chondroitinase  and hyaluronidase  solution at 37 °C for COL2 and MMP‐13, and by heating the sections bathed in sodium citrate solution  at 90 °C for the other target proteins. The tissue sections were counter‐stained with Vector Hematoxylin QS (for immunohistochemistry) or 4′,6‐diamidino‐2‐phenylindole  supplied by Vector Laboratories . The stained samples were imaged on an Olympus IX81 inverted microscope  or a Nikon Eclipse E800 upright microscope .OC and SFT microtissues were formalin‐fixed, paraffin‐embedded , and 6 µm thick sections prepared. To assess tissue phenotypes, OC sections were incubated in antibodies against OCN  was introduced in the FM stream to challenge the SFT for 1 week  miniJoint served as the control group.After confirming tissue phenotypes in the healthy miniJoint chips, the proinflammatory cytokine IL‐1neration.57 Aftβ through the SFT microtissue to the SM stream. To examine this potential issue and further confirm the role of SFT inflammation in inducing OC‐C degeneration, normal or acellular SFT (GelMA scaffold only) inserts were used in the miniJoint and subjected to IL‐1β treatment. After 1 week of culture in the chip, IL‐1β (10 ng mL−1) was introduced in the FM of chips with cell‐laden or cell‐free SFT and lasted 7 days. Chips with cell‐laden SFT receiving normal FM (without IL‐1β addition) served as the control. The OC‐C microtissues from the three groups were collected for transcriptomic analysis.One concern was cartilage degeneration caused by the diffusion of IL‐1The establishment of healthy and inflammatory miniJoint models was repeated two times and similar results were obtained.The transcriptome differences among the tissues from normal and inflamed miniJoint chips were examined by RNA‐Seq. Microtissues were lysed with the QIAzol reagent (Qiagen), and RNA was isolated from the lysate using an RNeasy Plus Universal Kit (Qiagen). Library preparation, sequencing, and bioinformatics analysis were performed according to Medgenome . Total mRNA was processed for next‐generation sequencing performed with the Illumina HiSeq 2500 system . Clontech SMARTer UltraTM Low Input RNA Kits  and NexteraXT  kits were used for library preparation. Low‐quality reads and adapter sequences were filtered out by tool Trimmomatic. Surviving reads were then aligned to human reference genome hg38 by Hisat2 aligner and gene counts were quantified by HTSeq. Differential expression analysis were performed based on gene counts by R package “DESeq2” and DEGs were selected by p‐value < = 0.05 and fold change > = 1.5. These DEGs were then applied to Ingenuity Pathway Analysis (IPA) to detect enriched pathways. This software includes databases of prebuilt pathways with known genes summarized from previous studies, and checks the overlap between the DEG list and known pathways and performs statistical tests to determine the enrichment. Significant pathways were defined by p‐value < = 0.05. Statistically stringently, FDR = 5% cutoff should be applied to control the false discovery rate. In order to encourage more gene candidates, this study went by p‐value< = 0.05 and fold‐change> = 1.5 cutoff. The numbers of DEGs defined by p‐value cutoff are shown in the corresponding Supporting Information Tables. All the tools were run by default parameter settings.Quality control was first applied on raw RNA sequencing reads by tool FastQC.58 Low gene counts across 18 normal and 20 OA human knee cartilage tissues were downloaded and compared. For human study GSE57218, genome‐wide gene expression profiles were collected across 7 healthy donors and 33 OA tissues. For the mouse study GSE143447, transcriptome expressions were quantified from 3 mice in the OA group that underwent surgical DMM and 3 mice in the sham‐operated control group. Within each study, similar differential expression analyses were performed as the miniJoint RNA‐Seq data. Gene counts were analyzed by R package “DESeq2” and bead‐chip data were analyzed by R package “limma.” Functional pathways were identified based on the DEGs, and common significant pathways were compared across the four studies. Network plot for DEGs in a selected pathway was drawn by the software Cytoscape.Gene expression profiles for two human OA patient studies and one mouse study were explored. For human study GSE114007,15 genComponents of the SM, including tissue‐specific secretome, proinflammatory cytokines, and degenerative enzymes, were measured using the Luminex multiplex immunoassay. Eighteen hours before harvesting the microtissues, all medium streams were carefully flushed with ≈30 mL rinsing medium  to remove the residual miniJoint culture medium. The tissues were then incubated in the rinsing medium for 18 h. Afterward, the conditioned medium was centrifuged at 14 000 g for 10 min before flash freezing the supernatant in liquid nitrogen. Luminex assays were carried out using the Bio‐Plex 200 system (Bio‐Rad). The Bio‐Plex Manager 6.1 software was used for data collection and analysis. The analyte panels used in this study were either commercial or custom‐designed products. The specific analytes and vendor information of each panel are provided in Table β treatment of the SFT, naproxen sodium  was added at 5 × 10−6 m to all medium streams and remained for 4 days. IL‐1β treatment continued for another 4 days as well . The IL‐1β challenged miniJoint without naproxen treatment served as the control. The microtissues and SM were collected for the various analysis described above.After a 3‐day IL‐1−1; Abcam), SOST protein , SM04690 , and IL‐1RA . To mimic the intraarticular use of these drugs, they were added only to SM and remained for 4 days in the inflamed miniJoint. IL‐1β treatment of the SFT continued for another 4 days as well .The utility of the miniJoint in drug screening was also assessed by introducing four potential DMOADs, including FGF18 , ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001).Each experiment was carried out with at least 3 biological replicates. Data is presented as mean ± standard deviation unless otherwise specified. Detailed information of sample size, pre‐processing, and statistical methods were specified in each figure legend. Prism 9  was used for statistical analysis. Significance level was set at 0.05 and indicated by * (The authors declare no conflict of interest.Z.L., R.S.T., and H.L. conceived and designed the experiments; Z.L., Z.L., H.Y., X.Z., L.Y., M.R.‐L., C.R., M.J.M, I.Y., E.N.L., M.R.F, Q.G., K.B.G., B.O., T.H., and B.M. carried out the experiments and analyzed the data; S.L., P.G.A., J.P.F., S.B.G., B.A.B., R.S.T., and H.L. performed the data analysis; Z.L. and H.L. wrote the first draft of manuscript; all authors edited and approved the manuscript; R.S.T. and H.L. obtained the funding.Supporting informationClick here for additional data file.Supplemental Video 1Click here for additional data file.Supplemental Table 1Click here for additional data file.