The widespread availability of antiretroviral therapy (ART) for people living with HIV (PLWH) has dramatically reduced mortality and improved life expectancy. However, even with suppression of HIV-1 replication, chronic immune activation and elevated inflammation persist. Chronic immune activation has been linked to a pro-inflammatory gut microbiome composition, exacerbated by compromised intestinal barrier integrity that occurs after HIV infection. Individuals living in urban versus rural areas of sub-Saharan Africa have differences in environmental factors such as water source or diet that may impact gut microbiome composition, yet immune phenotype and gut microbiome composition response to ART in PLWH living in rural versus urban areas of sub-Saharan Africa have not been compared. Here, we measured immune phenotypes and fecal microbiome composition in PLWH and healthy participants recruited from the urban Mabvuku polyclinic in the city of Harare, Zimbabwe and the Mutoko District hospital located in a district 146 km from Harare that services surrounding rural villages. PLWH were either ART naïve at baseline and sampled again after 24 weeks of treatment with efavirenz/lamivudine/tenofovir disoproxil fumarate (EFV/3TC/TDF) and the prophylactic antibiotic cotrimoxazole or were ART experienced at both timepoints. Although expected reductions in the inflammatory marker IL-6, T-cell activation, and exhaustion were observed in individuals who had suppressed HIV-1 with treatment, these changes were significant only when considering individuals in the urban and not the rural area. Gut microbiome composition showed more marked differences from healthy controls in the ART experienced compared to ART naïve cohort, and consistent longitudinal changes were also observed in ART naïve PLWH after 24 weeks of treatment, including a reduction in alpha diversity and altered composition. However, gut microbiome composition showed a more pronounced relationship with chronic immune activation and exhaustion phenotypes in the ART naïve compared to ART experienced PLWH, suggesting a particularly significant role for the gut microbiome in disease progression in uncontrolled infection. Human Immunodeficiency Virus 1 (HIV-1) infection is characterized by progressive infection and depletion of CD4 + T cells, chronic immune activation, and immune exhaustion that predisposes the infected individual to opportunistic infections and cancers defining acquired immunodeficiency syndrome (AIDS). Antiretroviral therapy (ART) has dramatically improved health outcomes in people living with HIV infection (PLWH). However, ART coverage remains suboptimal in many parts of the developing world, including in sub-Saharan Africa (SSA) where about 70% of the global HIV epidemic is concentrated . Even wiin vivo and to drive higher T cell activation in vitro [ex vivo [Factors that can drive chronic immune activation in PLWH on effective ART include co-infections and persistent antigen stimulation from residual viremia or the intestinal microbiome . This coin vitro  and cyto[ex vivo . Relativ[ex vivo –15, and [ex vivo . Second,[ex vivo . Finally[ex vivo .Individuals living near urban centers versus in rural areas of sub-Saharan Africa have differences in environmental exposures such as water source or diet that may impact their gut microbiome composition and immune response to microbes , 20. TheWe recruited 162 individuals with approximately equal numbers from the urban Mabvuku Polyclinic in the city of Harare and the Mutoko District Hospital, located in a district of around 161,000 people  146 km fClinical and demographic characteristics of study population by cohort at baseline visit. P-values were calculated using the Mann Whitney U test. Virologic failure is defined as PLWH who are on ART but have uncontrolled viral replication(> 200 copies of HIV-1 RNA/mL of plasma) , 25. BMICohorts had similar proportions of females versus males . ART naïve PLWH and HC had a similar median age of 35 and 36 respectively and ART experienced PLWH were significantly older (median age of 45). Median Body Mass Index (BMI) was in the normal range for the overall population (BMI = 22) and HCs had higher BMI compared to both ART Naïve and experienced PLWH in the rural (24 versus 22) but not the urban area (Table S1). Women had a significantly higher BMI than men, with more men (~ 11–17%) than women (~ 2%) in the underweight categories (Table S2). The median duration of ART and cotrimoxazole treatment among the ART experienced cohort at baseline was 89 and 84 months respectively. The median duration of cotrimoxazole treatment at baseline was 1 day (interquartile range (IQR) 0–6.6 days) in the ART naïve cohort , but sigAs expected, the ART naïve cohort had higher HIV viral loads and lower CD4 + T cell count, CD4 + T cell percent, and CD4/CD8 T cell ratio compared to the ART experienced cohort at baseline  and 7 1. Levels We next used flow cytometry to gain a deeper understanding of the effects of successful ART on CD4 + and CD8 + T-cell populations in blood. The immune characterization included: 1) chronic immune activation using HLA-DR and CD38 as markers, a common measure of disease progression in studies of HIV –32; 2) aAs expected, ART naïve PLWH had higher levels of chronically activated (CD38 + HLA-DR+) and exhausted (PD1+) CD4 + and CD8 + T cells compared to HC -targeted sequencing of fecal samples. One potential confounder in understanding the effects of ART on the microbiome in PLWH is the concomitant use of the antibiotic cotrimoxazole. There was also some exposure of ART naïve individuals to cotrimoxazole at baseline, and this was greater in people living in the rural versus urban location . DespiteThe ART experienced cohort had the lowest alpha diversity at baseline in both locations . ART naiPrevotella (17.71% mean relative abundance +/− 13.07%) and low Bacteroides (9.42% mean relative abundance +/− 9.26%) . In a principal coordinate analysis (PCoA) of weighted UniFrac [Prevotella and Bacteroides genera , the ART naïve versus HC comparison additionally showed decreases in Turicibacter, Blastocystis and Butyrivibrio .Comparisons between ART experienced PLWH and HC baseline values showed an increase in tricto_1 . In modedetected . Finallydetected  to detecTo gain further insight into relationships between gut microbiome composition and immune phenotypes, we used linear models with the immune markers described in For microbiome explanatory variables, we found that in ART naïve individuals only, the inflammatory marker IL-6 was negatively associated with PC3 and positively associated with PC4 of the weighted UniFrac PCoA analysis shown in Other interesting observations for non-microbiome explanatory variables included that BMI negatively correlated with CD4 + HLADR + CD38 + T cells in ART naïve PLWH only, consistent with a relationship between wasting and disease progression in untreated HIV infection, and positively correlated with CRP in HCs only, which is consistent with previously described relationships between CRP and obesity . Living Each row within each square represents one model. Circles represent predictors included in models. Predictors were determined by backwards stepwise regression feature selection. Location was retained in all models, viral load in the 2 PLWH cohorts, and HIV diagnosis date in the ART experienced cohort. Predictors that had a significant impact on the model are colored red (↑/ positive) or blue (↓/negative). Gender (up) represents higher values in males; location (up) represents higher values in the rural location; BMI categories (up) represents higher values with higher BMI; water source (up) represents higher values in people who drink from wells as opposed to tap; education level (up) represents higher values in people who went to secondary school as compared to tertiary; manual job (up) represents higher values in those who do work manual jobs as compared to those who don’t. “Model NS” means that the overall model was not significant despite the predictor being significant. Grey circles represent non-significant predictors.We next used linear regression to identify individual microbes and modules of highly co-correlated microbes  and a negative relationship with an ASV in the order Oscillospirales: UCG-010 , a positive relationship between an ASV in the genus Gastranaerophiliales with CD4 + PD1 + T cells at baseline  and a negative association between an ASV in the Eubacterium siraeum group with CD4 + CD103 + T cells at baseline .To evaluate microbe-immune relationships specific to treated infection versus HCs, we used a similar approach to identify relationships between ASVs and immune phenotypes that were significant in ART experienced PLWH but not HCs see . These tAlthough substantial gains in ART coverage for PLWH in SSA have led to improvements in life expectancy and reduced mortality , adherenAs expected, ART naïve PLWH had lower CD4 + T cell percentages and CD4+/CD8 + T cell ratios. We observed significantly higher CD4 + T cell percent and CD4/CD8 T cell ratio in ART naive women compared to men, which is consistent with the results of a prior study conducted in Nigeria . ART naïWhen restricting our subsequent analyses to only individuals who achieved viremic control, we observed significant reductions in levels of chronic T cell activation and exhaustion with ART. Interestingly, in stratified analyses, we found that these reductions were mostly significant only when considering individuals from the urban and not the rural location. This was the case both when comparing individuals who were ART naïve versus experienced at baseline and when looking at longitudinal changes that occurred before and after 24 weeks of ART. Prior studies have described significant challenges in administering ART in rural areas of SSA, such as longer travel distances to access health care facilities, increased stigma, and human resources challenges , 22, 59.One surprising result of our study was that individuals in our HC cohort and the cohort of people on effective ART at both timepoints had some worsening of immune phenotypes over time. For instance, the HC had a significant increase in CD8 + PD1 + T cells. Additionally, when stratified by location, the ART experienced cohort in the rural location had an increase in the inflammatory marker CRP over time . One potSince one goal of this study was to relate immune markers in HIV to the intestinal microbiome, we were also interested in evaluating T cells expressing the mucosal trafficking marker CD103. The chief ligand for CD103 is E-cadherin, a cellular adhesion molecule found on epithelial cells which is important for T cell homing to mucosal sites, including the intestine . CirculaOur study population overall had a relatively Prevotella-rich/Bacteroides-poor microbiome, which is consistent with other studies conducted in SSA , 63, 64.Overall, differences with untreated HIV infection were not pronounced, as has been indicated previously . HIV infBacteroides massiliensis [Prevotella massiliensis [Erysipelatoclostridium, a genus containing Thomasclavelia ramosa  [Bacteroides massiliensis is additionally a mucin degrader with a strong preference for host mucin glycans over dietary substrates [Prevotella massiliensis has previously been found to correlate with CD8 + CD38 + HLA-DR + cells in ART experienced HIV positive MSM [Parabacteroides [Coprococcus [Coprococcus has been associated with improved barrier function among ART experience HIV positive MSM previously [in vitro stimulations of Peripheral Blood Mononuclear Cells (PBMCs) despite a lack of pronounced microbiome differences compared to HCs [in vitro stimulation correlated with ex vivo levels in matched patient blood, supporting clinical significance [Despite a lack of pronounced microbiome differences in ART naïve PLWH, we did find significant relationships between gut microbiome composition and inflammation. Specifically, an association between PC3 and PC4 of the weighted UniFrac PCoA analysis and levels of IL-6 was observed in the ART naïve cohort in linear models. Furthermore, when using pairwise linear regression and network analysis to identify ASVs that were correlated with immune phenotypes Specifically in the ART naïve PLWH, CD4 + PD1 + was the strongest hub, followed by CD8 + CD38 + HLA-DR + T cells. ASVs that were positively correlated with these cell populations included bacteria that have been associated with bacteremia, intra-abdominal infections, colorectal cancer and/or inflammatory bowel diseases including iliensis , Prevoteiliensis , Erysiperamosum) , and Sutramosum) , 72. Bacbstrates , an actibstrates . Prevotetive MSM . Many ofteroides , and Coprococcus . Coprocoeviously  and is keviously . Taken td to HCs . These lificance .The ART experienced cohort had more pronounced microbiome differences from the HCs compared to ART naïve PLWH. ART was associated with lower alpha diversity in both cross-sectional comparisons and longitudinal analysis of the rural site. ART status also had a significant relationship with beta diversity, and more taxa significantly differed between the ART experienced cohorts and HCs than between ART naïve and HCs. Since ART treatment was coupled with use of the prophylactic antibiotic cotrimoxazole, it is difficult to disentangle the effects of ART drugs from the effects of the antibiotic. Our results suggest some influence of cotrimoxazole, since dysbiosis  correlated positively with length on cotrimoxazole in the ART naïve PLWH at baseline. This is consistent with cotrimoxazole being associated with changes in composition but not a loss of alpha diversity in a placebo-controlled trial . In anotReduced alpha diversity with ART plus cotrimoxazole compared to ART naïve PLWH is consistent with other studies evaluating PLWH exposed to both ART and cotrimoxazole  and to PMegamonas, Bilophila, and Lachnoclostridium, which have all been found to be increased in various prior studies of microbiome differences with treated HIV [Megamonas has additionally been associated with iron deficiency in the context of treated HIV infection in resource limited settings [Bilophila and Megamonas can be pro-inflammatory [Turicibacter, Butyrivibrio, Blastocystis, and other poorly defined ASVs in the order Clostridiales. Despite the possibility for microbiome differences that are observed with ART to be involved in inflammation, we found fewer correlations between immune phenotypes and the microbiome within ART experienced PLWH. Linear models indicated that microbiome differences not correlated with ART, namely microbiomes that differentiated by Prevotella versus Bacteroides ASVs (PC1 axis), were related to levels of immune exhaustion in treated infection. Linear models used to identify relationships between individual microbes and immune populations in ART experienced PLWH identified only a few correlations, again with immune exhaustion. These included associations between CD4 + PD1 + T cells and Gastranaerophilales (positive), Oscillospirales (negative), and Enterobacteriaceae (positive).Taxa that were significantly enriched in ART experienced PLWH compared to HC include ated HIV , 15, 84;ammatory . Taxa thOur study does have some weaknesses. Since we only used 16S rRNA targeted sequencing of bacteria to assess microbiome composition, our results do not rule out a role for fungi, parasites, viruses, strain level variation or differences in expressed functions. Furthermore, although prior work in our laboratory has been able to validate immune modulation by fecal bacterial communities using in vitro assays or gnotobiotic mice , we collWe found that although the percent of individuals who achieved viral control with ART was not different between PLWH in rural versus urban Zimbabwe, immunologic improvements with successful ART were muted in the rural compared to the urban area for inflammatory, T cell activation and T cell exhaustion phenotypes. More work is needed to determine underlying factors that may drive these differences. We found that ART with cotrimoxazole prophylaxis had a relatively strong impact on gut microbiome composition compared to HIV infection alone, with reductions in alpha diversity and greater deviation from the microbiome composition of HCs. Furthermore, we observed significant relationships between gut microbiome composition and inflammation, T cell exhaustion and chronic immune activation markers particularly in ART naïve PLWH, but also in the context of treated infection.The rural recruitment site was the Mutoko District Hospital, which is in a small town  about a 2 hour drive from the city of Harare, that services surrounding rural villages. The urban subjects were recruited from the Mabvuku Polyclinic, a large urban clinic administered by the City of Harare. Subjects were excluded from all cohorts if they had used antibiotics (apart from co-trimoxazole) within the prior two months, were pregnant, or had a Body Mass Index (BMI) greater than 29 kg/m2(are obese). All participants were 18 years old or older.At the first visit after informed consent was obtained, study participants were given a fecal collection kit. Stool samples were collected in a specimen collector within 24 hours prior to their second and third clinic visits, and aliquoted by the study participant into an OmniGene Gut collection system  for preservation of DNA. A fasting blood sample was collected by venipuncture during the second and third visits. Blood and fecal samples from the rural clinic were couriered to the Infectious Diseases Research Laboratory in the Internal Medicine Unit at the University of Zimbabwe Faculty of Medicine and Health Sciences (Harare), which is a 2 hour drive from the Mutoko District Hospital, in a cooler on the day of sample collection and then stored at −80°C. Samples for microbiome sequencing were shipped on dry ice to the University of Colorado Anschutz Medical Campus  and stored at −80°C upon arrival.Study participants filled out a questionnaire which included questions on age, Body Mass Index (BMI), gender, education level, water source, and factors related to occupation and home life.A subset of the blood sample was analyzed by flow cytometry at the Infectious Diseases Research Laboratory (IDRL) located in the Internal Medicine Unit at the University of Zimbabwe Faculty of Medicine and Health Sciences. Whole blood was collected in BD Vacutainer tubes containing sodium heparin and red blood cells (RBCs) from 500 μL of blood were lysed with 1x RBC lysis Buffer (Thermo Fisher). Cells were washed twice with staining buffer containing PBS, 2% BSA, 1mM EDTA and surface stained with BV785-labelled anti-CD3 antibody (BioLegend Cat# 317330), PerCP/Cy5.5-labelled anti-CD4 (BioLegend Cat# 317428), BV421-labelled anti-CD8 antibody (BioLegend Cat# 344748), BV605 labelled anti-CD38 antibody (BioLegend Cat# 303533), Pe-labelled anti-CD103 antibody (BioLegend Cat# 350206), Pe-Cy7 labelled anti-HLA-DR antibody (BioLegend Cat# 307616) and FITC-labelled anti-PD-1 antibody (BioLegend Cat# 329904) or appropriate fluorescence minus one (FMO) controls. Cells were washed twice with staining buffer and fixed in 1% formaldehyde. Cells were acquired on a BD LSRFortessa Flow Cytometer and analyzed by FlowJo.Plasma was isolated and frozen for shipment to the University of Colorado for measurement of CRP and IL-6 with ELISA following the manufacturer’s protocol . Blood samples were also used to evaluate absolute CD4 + T cell count or CD4 + T cell percent using the Sysmex  CyFLOW Cytometer, and CD4 easy count kit or CD4 percent easy count kit following manufacturer’s instructions , and HIV viral load using a Roche COBAS AmpliPrep/COBAS TaqMan (CAP/CTM) instrument with COBAS AmpliPrep/TaqMan HIV-1 test v2.0 kit following manufacturer’s instructions at the Infectious Diseases Research Laboratory (IDRL) located in the Internal Medicine Unit, UZ Faculty of Medicine and Health Sciences (UZFMHS).DNA was extracted using the DNeasy PowerSoil Kit protocol (Qiagen). Extracted DNA was PCR amplified with barcoded primers targeting the V4 region of 16S rRNA gene according to the Earth Microbiome Project 16S Illumina Amplicon protocol with the 515F:806R primer constructs . A steriUnless otherwise specified, all analyses were run in R version 4.2.2 (2022–10-31) .Before analyses were performed one sample with no immune data (ZIM033.3) was removed. ART naïve patients at week 24 and ART experienced patients at week 0 were labelled as “viremic” if they had more than 200 copies of HIV/mL of blood . There wFixed-effects Ordinary Least Squares (OLS) Linear Modelingfeols function in the fixest package [Linear modeling performed in Figure S3  package  in R to ggplot [Model . ggplot  was thenggplot [For the inter-cohort tests , all basggplot  was usedDemultiplexing of 16S rDNA gene sequences and quality control using DADA2  to definlm function in stats package [Alpha diversity, measured as Shannon entropy , was plo package .(M2)Δlm function in stats package [Beta diversity, measured using weighted UniFrac distances , was cal package  for eachWeighted UniFrac PCoA results from the core metrics analysis were used to calculate coordinates for biplots in QIIME2 and then visualized in R. To evaluate potential moderators of beta diversity two different Adonis tests  were perBoth models M4 and M5 were stratified (using the strata parameter) by PID to account for the dependence between samples from the same patient. Age was included in model M5 as a potential confounder since it significantly differed across cohorts.To determine genera that significantly differed in abundance across cohorts, we used ANCOM-BC (version 2.0.2) . This alModel M6 was run only on baseline values. Also, values were grouped by cohort which is used to help detect structural zeros and performing multi-group comparisons. Results are shown in Model M7 evaluated values at both time points (both week 0 and week 24) and grouped by cohort as in model M6. The random effects term was added to account for the dependence between samples of the same patient. Results are shown in ggplot [microViz [Model M8 evaluated values at both time points but only for those patients who were ART naïve PLWH at baseline, to evaluate changes over time with ART. No results were visualized for this model as there were very few differences. For models M6 and M7, visualizations ,B were pggplot  and micrmicroViz .lm function from stats package [To understand the impact that other variables might have on patient immunity  we curat package .Data for 8 immune markers and 237 microbial features were combined in a dataset spanning 127 participants at baseline and 113 participants at Week 24. Analyses were run separately for baseline and week 24 data. Microbial features were limited to those observed in > 20% of samples, resulting in 191 single ASVs and 46 modules of highly-co-correlated ASVs aggregated by SCNIC using default parameters . Featureigraph [The read count term accounted for differences in across samples since this analysis did not use rarefied data. To identify relationships that were evident in ART naïve PLWH and different from ART experienced PLWH or HCs, resulting models were filtered to include only those with an FDR-adjusted p-value on the F statistic of the overall regression < 0.2, adjusted R2 > 0.25, p-value on the slope for the ART naïve cohort < 0.05 and different from the slope for the ART experienced cohort and/or the HCs (p < 0.05 for at least one of these slopes), and maximum absolute value of DFFITS < 2 (to exclude results that were outlier driven). The resulting network and models were visualized using the VOLARE  web appligraph  library


The harm caused by cigarette smoking is overwhelmingly due to byproducts of tobacco combustion. Electronic Nicotine Delivery Systems (ENDS) provide nicotine to users without combustion, and may support tobacco harm reduction among cigarette smokers who would not otherwise quit in the near term. Analyses of Wave 5 of the Population Assessment of Tobacco and Health (PATH) Study compared biomarkers of exposure (BOE) levels for nicotine, 3 metals, 2 tobacco-specific nitrosamines and 14 smoking-related volatile organic compounds in 151 exclusive ENDS users, 1341 exclusive cigarette smokers, 115 dual users (cigarettes and ENDS), and 1846 past 30-day nonusers of tobacco, adjusting for demographics. Nicotine exposure in ENDS users and dual users did not significantly differ from smokers. Among ENDS users, 16 of 18 other BOEs were significantly lower than smokers’; 9 BOEs were not significantly different from nonusers. Among dual users smoking < 10 cigarettes/day, 15 of 18 non-nicotine BOEs were significantly lower than smokers’, whereas in dual users smoking ≥ 10 cigarettes per day none of the BOEs significantly differed from smokers’. In this representative sample of US adults, exclusive use of ENDS (vs. cigarette smoking) was associated with much lower exposures to many harmful chemicals associated with smoking-related disease. BOE levels in dual users were directly related to their cigarette consumption. These BOE data provide further evidence that ENDS expose users to substantially lower levels of toxicants than combustible cigarettes, confirming their potential for harm reduction. The harm caused by cigarette smoking is primarily due to exposure to byproducts of tobacco combustion4: cigarette smoke contains more than 7000 harmful chemicals including nearly 70 carcinogens. The US Food and Drug Administration has identified harmful and potentially harmful constituents (HPHCs) in cigarette smoke and in other tobacco products that are known to cause cancer and cardiovascular and respiratory diseases6. The risk and severity of many smoking-induced diseases are directly related to the level of exposure to the HPHCs in cigarette smoke5; hence reductions in exposure to HPHCs are expected to reduce risk for these diseases and many other adverse health effects8.Cigarette smoking is responsible for more than seven million deaths annually worldwide8. BOEs provide a measure of the actual human HPHC absorption associated with tobacco use, and thus can help quantify the potential health risks of tobacco products8.Many HPHCs are not directly measurable in the bodies of smokers; however, exposures can be assessed and quantified by measuring biomarkers of exposure (BOEs)—metabolites of the HPHC that are detectable in the urine and bloodstream9, and thus do not expose users to combustion byproducts such as carbon monoxide (CO) and polycyclic aromatic hydrocarbons11. Clinical trials demonstrate that smokers who switch to exclusive ENDS use experience significant reductions in tobacco specific nitrosamines (TSNAs), such as NNAL—a biomarker for NNK, and known carcinogen12.In contrast to cigarettes, electronic nicotine delivery systems (ENDS) deliver nicotine without combusting tobacco13. For example, acrolein (whose exposure can be assessed using 3-hydroxypropyl-mercapturic acid [3-HPMA]) is a respiratory irritant that is also associated with increased risk of cardiovascular disease14, and 1,3-butadiene (whose exposure can be assessed using monohydroxylbutenyl-mercapturic acid [MHBMA]) is a known human carcinogen and is also associated with respiratory and reproductive toxicity17. Additionally, some studies suggest metals can leech from heating coils into ENDS aerosols19, potentially causing adverse health effects given their high level of toxicity and carcinogenic effects21.However, there is a need to establish whether, like cigarettes, ENDS expose users to volatile organic compounds (VOCs)—HPHCs that exhibit dose–response relationships with cancer and cardiovascular disease risk23, and that among dual users, it is cigarette smoking, and not ENDS use, that is the primary driver of HPHC exposures26. Consistent with these findings, two longitudinal studies using data from PATH Wave 1 (2013–2014) to Wave 2 (2014–2015) found that smokers who switch from cigarette smoking to exclusive ENDS use experienced substantial decreases in BOEs, and smokers who transition to dual use and reduce their cigarette consumption by at least 50% also experience significant reductions in BOEs28.Several recent analyses have utilized BOE data from the Population Assessment of Tobacco and Health (PATH) Study, a longitudinal cohort study of tobacco use among a nationally representative sample of US adults, to assess variation in HPHC exposure. Cross-sectional analyses of PATH Wave 1 (2013–2014) demonstrated that exclusive ENDS users have lower levels of many of the measured BOEs compared to cigarette smokers and dual users31. These later-generation products deliver nicotine more efficiently than earlier-generation products33, and some implement better control of the temperature to which the e-liquid is heated, a major factor in production of toxic compounds34; hence there is a need to assess differences in BOEs among ENDS users in more recently-collected data.Although these studies offer valuable insights into HPHC exposures among ENDS users, the findings are limited to early-generation ENDS products marketed when the data was collected between 2013–2015. ENDS products have rapidly evolved since 2015, with open system modular devices and fourth-generation pod-based nicotine-salt ENDS products becoming increasingly prevalent35, exposures among dual users were also evaluated by their level of cigarette consumption.The primary aim of the current study was to assess differences in levels of BOEs to TSNAs, VOCs, metals and nicotine, comparing: (i) exclusive ENDS users; (ii) exclusive smokers; (iii) dual users; and (iv) past 30-day tobacco non-users using data from Wave 5 (2018–2019) of the PATH Study. Further, given heterogeneity in cigarette smoking behaviors among dual users36. Recruitment used a stratified address-based, area-probability sampling design; survey weights are used to produce national estimates36. The study was conducted by Westat under a contract with the Food and Drug Administration (FDA) Center for Tobacco Products (CTP), National Institute on Drug Abuse (NIDA) and National Institutes of Health (NIH). Westat’s Institutional Review Board (IRB) approved the study design and data collection protocol36. All participants in the PATH Study provided informed consent36. This secondary analysis of deidentified PATH Study data was reviewed by Advarra IRB Number IRB00000971 and deemed exempt on the basis of 45 CFR 46.104(d)(4). Use of these data was approved by the Inter-university Consortium for Political and Social Research (ICPSR), which reviewed analytic results and cleared these results for public dissemination on the basis that they did not present a risk of identifying individual participants. All procedures were performed in accordance with relevant guidelines.The PATH Study is a nationally-representative, longitudinal cohort study of adults and youth in the US37 and Restricted-Use Files (RUF)38. Urine specimens were analyzed by the Centers for Disease Control and Prevention (CDC), National Center of Environmental Health. Because they contain more granular data which may pose a risk to respondent identification, the PATH Study BRUF and PATH Study RUF are only available to researchers that apply for access and complete a data use agreement, which includes review of statistical outputs before any results can be published. At this time, PATH Study biomarker data are available only in restricted-use files. Additional information is provided by36 and at 10.3886/ICPSR36840.v18 and 10.3886/ICPSR36231.v31.The current analyses used data from Wave 5 (December 2018 to November 2019) of the PATH Study Biomarker Restricted-Use Files (BRUF)The analytic sample for the current manuscript is comprised of PATH Study Wave 5 adult respondents who were at least 21 years old at time of interview, provided urine samples and also had (i) valid data on sex and race/ethnicity; (ii) body mass index (BMI) between 18–40; (iii) urinary creatinine in the range 10–370 mg/dL; and (iv) valid BOE data on one or more analyte (including imputed data below the limit of detection [LOD]). To avoid confounding of BOEs by other exposures, participants were excluded if they reported past 30-day use of other tobacco, nicotine, or marijuana products  at interview or past 3-day use at urine collection Table . FurtherParticipants were initially classified into one of four groups on the basis of past 30-day ENDS use and past 30-day cigarette smoking: (i) exclusive ENDS users ; (ii) exclusive cigarette smokers ; (iii) dual users ; and (iv) past 30-day tobacco product nonusers . Preliminary analyses showed BOE levels were more heterogeneous in Dual Users than in other tobacco use groups Figs. , and S4.6. Altogether, 15 BOEs were considered to be relevant to cancer, 9 to respiratory disease, 8 to reproductive or developmental problems, and 4 to cardiovascular disease, with most BOEs relevant to multiple disease classes.In Version 18 of the PATH Study Biomarker Restricted-Use Files, available Wave 5 urine panels include BOE data for nicotine, metals, TSNAs, and VOCs. All 14 available VOC markers, two available TSNAs, and three available metals, as well as two aggregate measures of nicotine and its metabolites  were analyzed  was equal to one.Urinary biomarker measurements were adjusted for urinary creatinine by expressing the concentration of the BOE as a ratio to the concentration of creatinine. This is an often-used adjustment for dilution of the spot urine samples by waterup Table . PairwisAdjusted analyses of geometric mean creatinine-adjusted BOE levels with two Dual User strata are presented in Figs. All analyses were conducted using SAS© software  and were weighted to represent the 2013–2014 U.S. adult civilian, noninstitutionalized population of never, current, and recent former (within 1-year) tobacco users with the Wave 5 single-wave weights for the Wave 1 Biomarker Core. Hypothesis tests used the 0.05 significance level without adjustment for multiple comparisons. Variance estimation used Fay’s Balanced Repeated Replication method with Fay’s factor set to 0.3 and 100 replicate weights to account for the complex survey design structure of the PATH Study. Estimates are reported for which the relative standard error (RSE) is greater than 30% or for which the proportion of subjects with measurements below the LOD exceeded 40%.SE = 0.93]) than Smokers (mean = 13.8 [SE = 0.39]). When stratified by median cigarettes/day, Dual Users formed two distinct groups: those who smoked less than 10 cigarettes per day  averaged 3.5 cigarettes/day (SE = 0.5) and those who smoked at least 10 cigarettes per day  averaged 14.5 cigarettes/day (SE = 1.5). Past 30-day smoking and ENDS use behaviors differed in the two Dual User strata; the average 10 + CPD Dual User smoked nearly every day (29.5 of the past 30 days) and used ENDS on about half as many days (18.2 of the past 30 days), whereas < 10 CPD Dual Users used ENDS (24.0 of the past 30 days) more frequently than cigarettes (20.6 days of the past 30 days).On average, Smokers and Nonusers were older than Dual Users and ENDS Users Table . Nearly About three-quarters of ENDS Users used open systems and used ENDS on approximately 28 of the last 30 days, on average , lower exposures are likely to be associated with lower disease risk, implying that smokers who switch to ENDS would likely experience decreased disease risk6.The results of this analysis concord with previous observational studies using data from the PATH Study44. In the present analysis, levels of both TSNA and 12 of 13 VOC BOEs were significantly lower in dual users reporting smoking < 10 cigarettes/day than in exclusive cigarette smokers, though levels of all BOEs assessed were significantly greater in < 10 CPD Dual Users than in Nonusers.Dual Users were extremely heterogeneous in exposure, and partitioning by cigarette consumption showed that the key driver of toxic exposure was the quantity of cigarette consumption. Some analyses have reported that dual users of both ENDS and cigarettes have exposures as high or even higher than those seen in exclusive smokers25, with HPHC exposure increasing with increasing cigarette consumption45.Conversely, Dual Users smoking 10 or more cigarettes/day—whose average cigarette consumption was similar to that seen in Smokers (mean CPD = 14.5 vs. 13.8), showed levels of BOEs that were not significantly lower than Smokers’ levels but higher than ENDS Users and Nonusers. Thus, the results indicate that the BOE levels observed in Dual Users are largely attributable to Dual Users’ cigarette smoking. This dose–exposure association is consistent with previous analyses of the PATH Study showing that cigarette smoking is the primary driver of HPHC exposure among dual users53. These data regarding HPHC exposure among dual users are especially pertinent given that dual use is a common initial use pattern among smokers who adopt ENDS, although it often serves as a transitionary state on the pathway to complete switching away from smoking54, ultimately reducing exposures further.The findings regarding dual use are also consistent with controlled clinical confinement studies and randomized trials demonstrating that dual users who reduce their daily cigarette consumption by at least 50% significantly reduce their HPHC exposure compared to smokers who continue smoking as usual9. It is logical that the chemical composition of the ENDS aerosol would be predictive of the users’ ultimate exposure, as users only inhale chemicals present in the aerosol matrix. This suggests that chemical analyses of the aerosols can be used to infer human exposures, which is particularly important since some constituents that can be measured in the aerosols do not have validated biomarkers.These findings regarding actual human exposure are consistent with chemical analyses of aerosols produced by ENDS under conditions that mimic real-world use, which show that these TSNAs and VOCs are absent or present at levels much lower than in cigarette smoke56. However, many of these experiments have been criticized for using procedures that do not replicate real world patterns of ENDS use and other methodological limitations 57. The results of the current study demonstrate that, on average, ENDS Users are exposed to lower levels of cadmium than Smokers. Future naturalistic research is needed to assess levels of other metals beyond the three included in the PATH Study. Exposure to many VOCs can occur from sources other than tobacco use, and thus nonusers represent the benchmark for baseline levels of environmental exposures to such VOCs8. For example, acrylamide exposure can occur from many foods as well as from tobacco use59, and the levels observed in ENDS users were almost exactly equivalent to those in nonusers of tobacco. PMA, a marker for benzene exposure, was not elevated in Smokers (vs. Nonusers), and showed similar levels across all groups, suggesting that benzene exposure was occurring primarily from environmental sources, rather than tobacco product use.Chemical analyses of ENDS aerosols have found levels of metals above established toxicological standards61. Used as a harm-reduction strategy, ENDS are intended to draw smokers away from combustible cigarettes, which are the most harmful and addictive nicotine-delivery product. Consistent with these conceptual models of switching and nicotine delivery, data from observational studies62 and randomized trials64 support the concept that adequate levels of nicotine are likely necessary to facilitate switching away from smoking.All the nicotine-using groups  demonstrated similar levels of nicotine exposure, all significantly higher than those in tobacco product nonusers. This is consistent with the concept that ENDS are intended as alternative nicotine sources to allow smokers who are not quitting to maintain nicotine intake, while reducing exposure to HPHCs associated with cigarette smokingStrengths of the study include the representative sample of US adults, large overall sample size and use of a dataset that includes more recent-generation ENDS devices. A limitation of the current analysis was that its measures of exposure were limited to select TSNAs, VOCs, three heavy metals, and nicotine. Other HPHCs such as polycyclic aromatic hydrocarbons (PAHs) were not available from PATH Study Wave 5 at the time of analysis (Version 18). The BOEs currently available from the PATH Study also did not include markers for exposures that might be higher in ENDS users. Finally, the analysis was a cross-sectional, between-groups comparison, so it does not directly show a within-participants reduction in HPHCs among smokers who switch to ENDS.11. In concert with the results of randomized clinical trials and longitudinal studies, the current results indicate that smokers who switch to ENDS likely experience reductions in health risk. The data also indicate that exposure to TSNAs, VOCs and metals among dual users was primarily a function of their cigarette smoking, as dual users who were smoking fewer than 10 cigarettes a day also consistently showed lower exposures compared to smokers. However, the data make clear that complete switching, with no smoking at all, results in reduced exposures compared to dual use with low levels of smoking. Further research on other toxicants, and also on biomarkers of potential harm, such as markers of inflammation, can inform reductions in health risks likely to accompany switching from smoking cigarettes to ENDS.In summary, this nationally-representative analysis of a large sample of US adults found that exclusive users of ENDS have significantly and substantially lower exposures to a range of TSNAs, VOCs and one metal compared to smokers, with exposures among ENDS users often comparable to those seen in individuals not using any tobacco products at all. Such reductions in exposure have been taken as evidence of reductions in risk of smoking-induced diseaseSupplementary Information.


Intraventricular hemorrhage is characterized by blood leaking into the cerebral ventricles and mixing with cerebrospinal fluid. A standard treatment method involves inserting a passive drainage catheter, known as an external ventricular drain (EVD), into the ventricle. EVDs have common adverse complications, including the occlusion of the catheter, that may lead to permanent neural damage or even mortality. In order to prevent such complications, a novel dual-lumen catheter (IRRAflow®) utilizing an active fluid exchange mechanism has been recently developed. However, the fluid dynamics of the exchange system have not been investigated. In this study, convective flow in a three-dimensional cerebral lateral ventricle with an inserted catheter is evaluated using an in-house lattice-Boltzmann-based fluid–solid interaction solver. Different treatment conditions are simulated, including injection temperature and patient position. Thermal and gravitational effects on medication distribution are studied using a dye simulator based on a recently-introduced (pseudo)spectral convection–diffusion equation solver. The effects of injection temperature and patient position on catheter performance are presented and discussed in terms of hematoma irrigation, vortical structures, mixing, and medication volume distribution. Results suggest that cold-temperature injections can increase catheter efficacy in terms of dye distribution and irrigation potential, both of which can be further guided by patient positioning.The online version contains supplementary material available at 10.1007/s10439-022-03130-9. However, this procedure lacks a well-defined clinical protocol and must be performed at the physician’s discretion.19The majority of hemorrhagic strokes happen due to intracerebral (ICH) or subarachnoid hemorrhages (SAH).active fluid exchange). Several clinical studies have illustrated the ability of this dual-lumen catheter system to deliver tissue Plasminogen Activator  in a continuous manner while enabling quicker evacuation of blood in the ventricles while negating catheter occlusions41 (increasing EVD efficiency and decreasing complications52).In order to take advantage of irrigation while maintaining sterility, a novel dual-lumen self-irrigating catheter system has been recently developed . This dual-lumen catheter is connected to an intelligent digital cassette  and powered by a control unit with a user interface. The catheter is designed to prevent the issues observed with conventional EVD usage by delivering periodic irrigation with drainage and continuous pressure monitoring , this study investigates the fluid exchange system within the lateral cerebral ventricle at different fluid injection temperature and patient body position. The results of this study might provide insights into treatment procedures of active-irrigation catheters and, further, may contribute to improved patient care by elucidating optimized drug delivery patterns.Although case studies with this catheter system (IRRAflow®) have documented the effectiveness of such self-irrigating catheters in shortening hospitalization times with no catheter-related complications,12 This approach has been utilized for various fluid dynamics problems including those related to turbulent,14 multiphase,40 thermal,39 and bio-fluid46 flows.In order to simulate fluid flow, we used a lattice Boltzmann method (LBM) that combines discrete kinetic theory and modified molecular dynamics. Unlike conventional continuum-based methods, LBM is a mesoscopic method that behaves like a conservation equation solver for the corresponding macroscopic variables while providing a viable way for the inclusion of microscopic dynamics at the interfaces.47 In the present work, our model has two sets of distributions 9 with 19 discrete velocity vectors (D3Q19), given byIn LBM, a particle distribution function ensures that fluid particles move synchronously on a regular lattice. The function is chosen to satisfy conservation equations, where the fluid is Galilean invariant and isotropic.39; pressure 46). The buoyancy force density 31 asHere, 6 Three-dimensional (3D) rendered images of this geometry is acquired from a repository43 and subsequently separated using SOLIDWORKS®  to obtain the final form. MeshLab13 is used for smoothing the geometry and generating three-node triangular mesh elements .22 IBM regulates the force term 35 The Lagrangian-interface force exerted on the wall by the fluid 22 given by20 Such a procedure can be summarized by the following steps, where steps one through five are iterated until the boundary conditions are satisfied:Step 0: Step 1: Step 2: Step 3: Step 4: Step 5: The fluid and solid domains are coupled via a summation of all intermediate values of such forces over all iterations, i.e., 30 to those concerning biological flow dynamics.46 A particular advantage of using this immersed-boundary lattice Boltzmann solver for FSI simulations lies in the ability of LBM to easily incorporate other physical effects as right-hand-side forcing terms of Eq.  with one drainage and six injection ports  whose locations corresponds to those found in the IRRAflow® catheter. The tip is inserted 18 mm into the ventricular domain in order to ensure all ports are completely inside the ventricle.37 Ideally, the injected and drained volumes should be equal in order to prevent collapsing or possible bursting of the ventricle.48 The average velocity is calculated based on the injection volume, and spatially-uniform velocity profiles are prescribed at the injection and drainage sites during their corresponding operating time intervals. The onsets of these velocities are regularized-in-time by partition-of-unity-based functions to obtain appropriately smooth and physically-relevant simulations.We have employed the standard duty cycle of IRRAFlow® in all simulations: 20 s with three distinct time intervals corresponding to injection (1 s), observation (9 s), and drainage (10 s). The considered injection volume of 44 The brain cells can tolerate low temperatures 44 However, they start to develop ischemia at temperatures that are slightly elevated above normal body temperature 7 Therefore, injections can only be performed at temperatures colder than the normal body temperature To the best of our knowledge, there is no prescribed injection temperature for this procedure, although it should be within a realistic range so as to avoid neural injury.46 This scheme is applied to the outer boundaries of the fluid domain as well as to the catheter boundaries , whereas the fixed temperature condition 31Fixed temperature boundary conditions and a bounce-back scheme are applied for the fluid flow simulations. The bounce-back method assures no-slip and no-penetration conditions by reversing the outgoing velocity distribution functions.11 with a thickness corresponding to Simulations are performed in non-dimensional lattice units, where the governing equations are non-dimensionalized by four reference parameters: a density 32 For the case of an injection temperature of All simulations are performed using a uniform mesh with a lattice spacing of 4 i.e.,32 and where The evolution of a dye concentration At all other boundaries, including rigid walls 4 We note4 based on a Fourier continuation (FC) methodology5 for accurate Fourier series expansions of non-periodic functions. For a dimension Solution of Eqs. –17 is fa of Eqs. –17 can t4The resulting function k in Eq.  can be c4 Such time marching incurs linearly-scaling Courant-Friedrichs-Lewy (CFL) constraints on the timestep.5 Together with the FC-based spatial scheme, the overall solver enables high-order accuracy and a faithful preservation of the dispersion/diffusion characteristics of the underlying continuous operator . The latter property is particularly important for avoiding non-physical  diffusion in simulated dye concentrations.A high-order explicit time integration scheme is utilized to complete the numerical treatment of the advection–diffusion system.fluid blending in the ventricle using Eq.  and prone (lying on the stomach with the face downwards). Other body positions are not considered since they have been demonstrated to be clinically impractical.Simulated snapshots, at various times, of the velocity and vorticity fields inside the ventricle are presented in Figs. Every velocity magnitude image in Fig. mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimSnapshots of the time evolution of the vortex structures corresponding to each configuration is presented in Fig. fluid blending caused by the injected fluid via the space-averaged vorticity for each configuration. We observe that vorticity curves produced by all injections are similar at their onset. However, midway through each injection, the subsequent evolution of the average vorticity begins to differ drastically as a function of temperature. We note that the coldest injection results in up to twice as much blending than at room temperature. Following the end of injection  and drug delivery performance (via consideration of vortex structures and dye evolution) under different injection temperatures and patient positions.This work studies the thermal and postural effects on irrigation patterns and injected fluid mixing of a newly-developed self-irrigating catheter to utilize an active fluid exchange mechanism for treating intraventricular hemorrhages. Through robust numerical simulations, we have investigated clot irrigation potential  instead of face-down (prone) for better irrigation . Additionally, we have not considered chemical reaction between injected tPA solution and hematomas in our simulations. Our results only elucidate the path of diffusion and not the actual activated interaction between tPA and hematomas . The addition of such a reaction term to the governing model Eq.  is a subSome additional limitations should be mentioned. To the best of our knowledge, the properties of an intraventricular hematoma  are not well established and there is a dearth of literature. As such, multi-phase fluid models are not feasible. Hence, we have employed a single-phase model by assuming the hematoma is a uniform mixture of blood and cerebrospinal fluid, where medication immediately mixes with the hematoma at the injection port. Further research into the properties of a hematoma may one day enable consideration of fluid–fluid interactions; however, conclusions drawn from our study would still be reasonably accurate since increased thermal forces (lower injection temperatures) enhance irrigation and drug distribution in any case.via patient positioning. Our unique modeling approach may help facilitate rapid assessment of drug delivery performance before treatment. Indeed, the first two days are most critical for IVH patients, and this work has been motivated by the overarching goal of faithfully producing treatment-specific simulations towards enabling a priori catheter performance estimation within a few hours.Overall, this work demonstrates that both temperature and patient position are important factors in catheter efficacy. Results suggest that treatment administered at a high temperature difference Supplementary file2 (MP4 642 kb)Supplementary file3 (MP4 642 kb)Supplementary file4 (MP4 1620 kb)Supplementary file5 (MP4 1350 kb)Supplementary file6 (MP4 1946 kb)Supplementary file7 (MP4 1418 kb)Supplementary file8 (MP4 2563 kb)Below is the link to the electronic supplementary material.


We implemented a GSC monolayer system to investigate in situDNA/RNA-sequencing was performed on 56 glioblastoma tissues and 19 derived GSC cultures. Sensitivity to TMZ was screened across 66 GSC cultures. Viability readouts were related to clinical parameters of corresponding patients and whole-transcriptome data.Tumour DNA and RNA sequences revealed strong similarity to corresponding GSCs despite loss of neuronal and immune interactions. In vitro TMZ screening yielded three response categories which significantly correlated with patient survival, therewith providing more specific prediction than the binary MGMT marker. Transcriptome analysis identified 121 genes related to TMZ sensitivity of which 21were validated in external datasets.GSCs retain patient-unique hallmark gene expressions despite loss of their natural environment. Drug screening using GSCs predicted patient response to TMZ more specifically than MGMT status, while transcriptome analysis identified potential biomarkers for this response. GSC drug screening therefore provides a tool to improve drug development and precision medicine for glioblastoma. Over the past 5 decades, great efforts have been put towards development of new anti-cancer therapies for the incurable brain tumour GBM. Promising preclinical results have been obtained for a plethora of new drugs, however, these results have not translated into improved outcomes in patients. One reason for this translational disconnect is that preclinical research in the past made use of clonal GBM cell line-based model systems that do not accurately reflect the molecular and phenotypic characteristics of these aggressive tumours . The secIn keeping with these findings, we set up a drug-screening platform based on serum-free culture of low passage patient-derived GSCs in monolayers. Culture in 2D offers the advantage of rapid and straightforward assessment of drug sensitivity while preserving genomic stability and interpatient tumour subtype signatures . In vitrThe results of our retrospective study demonstrate the value of a simple and straightforward 2D assay on patient-derived GSCs for predicting clinical response to TMZ, despite the absence of the in situ microenvironment. Not only could responders and non-responders be identified but also an intermediate responding group, which offers a marked improvement to the binary classification by MGMT promoter methylation analysis. Moreover, our results suggest that this model may offer a tool in preclinical research and drug development to identify effective new tumour-targeted therapies for (subtypes of) GBM, as well as corresponding biomarkers of response. Taken together, our study provides support for further development of patient-derived GBM stem cell monolayer drug screening assays toward implementation in preclinical drug development programs as well as precision medicine approaches.GSC cultures were obtained from our biobank containing tissue and derived cell cultures from primary brain tumour samples from the Erasmus Medical Centre, Rotterdam and Elisabeth Tweesteden Hospital, Tilburg, both in the Netherlands. The use of patient tissue for this study was approved by the local ethics committees of these hospitals and all patients signed informed consent forms according to the guidelines of the Institutional Review Boards of the respective hospitals. Tumour classification was performed by the local neuropathologist according to guidelines of the WHO 2007 and 2016 classification of primary brain tumours. The biobank was searched for samples obtained between September 2009 and May 2019 matching the following criteria: primary glioblastoma (no recurrent), IDH wild type, fresh frozen tumour material available, successful GSC culture below passage 10 available, patient received standard treatment (radiotherapy with concomitant and adjuvant temozolomide ), and avAs primary endpoints for outcome, we used progression-free survival (PFS) and overall survival (OS). PFS was calculated from the day of operation to the first evidence of radiological and/or clinical progression, as concluded in radiology reports of follow-up MRI scans. OS was calculated from the day of operation until death.Fresh glioma tissue samples obtained directly from the operating room were processed according to protocols previously described to obtain monolayer GSC cultures under serum-free conditions , 18. DetTM kit  and used as a template for methylation specific (MS) PCR. Primers specific for methylated and unmethylated MGMT promoter DNA and PCR conditions were used as described previously  vs normalized response with variable slope model was applied to calculate the IC50 values. The reported IC50 is the concentration of TMZ that gives a response half way between the highest and lowest value of each curve. In some cases this value may deviate from Y = 50. Survival curves were generated using GraphPad Prism and survival analyses were performed using SPSS (IBM Statistics 28.0.1.0). Student’s t test was used to correlate in vitro sensitivity of TMZ with MGMT status. P values below 0.05 were considered significant for these analyses.GraphPad Prism software version 9 (GraphPad Software) was used for defining ICn = 19). Correlation analysis was performed to assess similarity between the paired samples. Results revealed that DNA sequences remained largely conserved between the tumours and their corresponding cell cultures as shown in Fig. To establish whether in vitro culture systems retain the genetic programs of matched tumours, total DNA and RNA was isolated and sequenced from a panel of tumours and derived cell cultures (p < 0.0001)  Fig. . The ind01) Fig. . As expe01) Fig. . GSEA of01) Fig. . Further01) Fig. . These t01) Fig. .Whether these changes affect the expression patterns of hallmark genes in GBM between tissues and derived cultures, was assessed by comparing expression levels of the hallmark genes EGFR, PDGFRA, PTEN, CDK4, RB1, TP53, MET, MYC, HIF1A, PIK3CA, STAT3 as well as MGMT Fig. . A complTaken together, our data implies that tumour samples undergo a transcriptomic shift from intercellular communication in situ to increased cell cycle and growth activity in vitro. Despite these changes, hallmark gene expression profiles are maintained in a patient-specific manner.To assess whether these changes in microenvironment affect the potential of ex vivo glioblastoma cultures to predict response to treatment, we retrieved tumour samples and derived GSC cultures from our glioma biobank for a retrospective study to correlate in vitro response to TMZ to clinical response. We identified 66 samples fulfilling the inclusion criteria of this study  the MGMT status was preserved. In 5 cases MGMT was unmethylated in the parental tumour, whereas it was found methylated in the derived cell culture . In 7 cases, MGMT promoter methylation in the tumour was lost in culture . A small percentage of samples revealed a selection from a mixed signal in the tumour to a homogeneous signal in culture (5%) Fig. .Fig. 2MG50 values, area under the curve (AUC) and percentage cell viability at 100 μM. This concentration is close to the maximum reported cerebrospinal fluid (CSF) concentration of 75 μM for TMZ and was previously found to differentiate between sensitive versus resistant GSC cultures to TMZ in our drug testing model [Dose-response analysis was performed upon screening of temozolomide on the panel of 66 primary GBM cultures , the IC50 values are based on extrapolation and are therefore less reliable. As a result, using the percentage cell viability and AUC values as readouts, yields more robust results which also show stronger correlation to MGMT status.As expected, MGMT methylated GSC cultures displayed higher sensitivity to TMZ than unmethylated cultures in all three readouts, with AUC revealing the largest difference Fig. . Contrarn = 11) were excluded from analysis to increase the uniformity of the cohort with regard to other prognostic factors such as age or Karnofsky Performance Score (KPS). For the remaining 55 patients, we performed Spearman correlation analysis and Cox regression between the in vitro response data  and their clinical outcome (PFS and OS). For the Spearman correlation analysis, % viability at 100 μM gave the strongest correlation with both PFS  and OS   Fig. .Fig. 3Inp = 0.03) and OS  whereas IC50 values revealed non-significant correlation with PFS  but significant correlation with OS   and OS (p < 0.00005)  . The outcome of responders and non-responders generally aligned with the cell culture MGMT status, however, for 33% of patients (the intermediates) cell culture MGMT status did not predict response to TMZ  did not significantly differentiate non-responders from intermediates but did discriminate responders from these groups   than between the MGMT levels and promoter methylation status (p = 0.011). Thus, compared to the established biomarker MGMT methylation status, which postulates a bimodal response to TMZ, our assay provides a means to separate patients more specifically into three distinct response categories.The distribution of the in vitro results revealed a gradient in TMZ response. We therefore visualized the correlation of in vitro response (%viability) vs. OS by Kaplan-Meier analysis using 3 groups to emphasize that the response is non-binary. Dividing the group by quartiles of % viability into responders (<50%), intermediate responders (50–75%) and non-responders (>75%) at 100 μM TMZ, revealed significantly different survival curves for the three groups  months for responders (R), intermediates (I) and non-responders (NR), respectively (log-rank R vs NR 11) Fig. . This seTMZ Fig. . The pat01) Fig. . For 19 To further substantiate these results, MRI images at follow-up time points post TMZ/RTx treatment were collected from 3 predicted responders (viability below 50%) and 3 predicted non-responders (viability above 75%). As shown in Fig. p = 0.0006) , of which n = 19 corresponding to the cell cultures, was correlated to the patients’ overall survival. Of note, all patients in the GLIOTRAIN cohort received TMZ + radiation therapy. This analysis revealed 2542 differentially-expressed genes which significantly correlated to survival, including MGMT  . Of the 2542 genes, 121 genes overlapped with the genes correlated to TMZ response in the in vitro data set  DNA repair, (ii) G2M check point, (iii) E2F targets (iv) PI3K/Akt/mTOR signalling and (v) myc targets  as well as to the publicly-available TCGA data considering only GBM patients who received TMZ treatment (n = 89). Within the GLIOTRAIN data set, 16 of 121 genes were significantly correlated to survival, including MGMT  (Table p = 0.025)  samples not included in the initial analysis (4) Table . Likewis5) Table . InteresIn this retrospective study we assessed whether loss of interactions between GBM cells and their tumour microenvironment affect the predictive value of a 2D functional screen testing a clinically-relevant pharmaceutical intervention. To do this, we charted the transcriptomic differences between GSC cultures and corresponding parental tumours, and we assessed the feasibility of the GSC monolayer screen to predict clinical efficacy of TMZ for GBM patients.Correlation analysis of DNA and RNA sequences from low-passage GBM cultures with parental tissues revealed in general a strong correlation between paired samples, including expression profiles for hallmark genes of GBM. However, transcriptomic comparison also revealed marked differences, with pathways related to neuronal intercellular communication and immune cell interactions being exclusive for tissues, and pathways related to cell cycle processes being upregulated in cell cultures. The origin of this transcriptomic shift can be attributed to the selection of cells from the in situ tumour environment that have an intrinsic survival advantage in vitro, as well as the adaptation of these cells to 2D culture condition. With the growing body of evidence that tumour micro-environmental interactions play an important role in GBM growth and development in situ –14, the To address this, we implemented a simple and straightforward 2D drug screening approach, which allowed accurate and reproducible drug efficacy assessment . This ofHOX signature, EGFR expression, as well as base excision repair enzymes have been linked to TMZ resistance [As expected, TMZ was more effective in MGMT methylated than unmethylated cell cultures Fig. , confirmsistance , 44. Altsistance .The use of patient-derived ex vivo tumour models for drug testing may thus aid in treatment selection. Such approaches are being undertaken for various types of cancer and the generated data confirm the predictive power of patient-derived in vitro assays, including GBM –49. HowaThe clinical relevance of TMZ testing in our GSC monolayer assay, also prompted us to investigate whether molecular markers related to TMZ sensitivity could also be identified using this system. The pathway analysis identified 10 pathways present in both cell culture and tumour data relating to TMZ response. Interestingly, many of these pathways have previously been reported to play a role in TMZ resistance mechanisms. DNA repair, G2/M checkpoint and E2F targets are closely related cellular processes and important players in TMZ resistance by regulating the cell cycle and allowing cells to repair genomic damage . DownstrA platform that can predict drug sensitivities of individual tumours may also advance the fulfilment of patient-tailored cancer treatment. Indeed, our data shows that interpatient variability in GBM hallmark gene expression levels is, in general, well-retained in the derived cell cultures. However, genomics-based targeted therapies have thus far not significantly improved outcome of GBM patients . One cauTaken together, despite a contextual shift in growing conditions from the natural tumour microenvironment to in vitro culture conditions, patient-derived GBM 2D ex vivo models offer a valuable tool in preclinical research and functional drug screening may aid in identifying effective therapies for subgroups or individual patients. With the implementation of such model systems in drug development programs, it is expected that promising agents will also perform better in (stratified) clinical trials, therewith improving the outlook for GBM patients.Supplemental FiguresSupplemental Tables 1–6Supplemental file 1Supplemental legends for Figures and TablesSupplemental Methods


This comparative study was conducted to assess the intramedullary bone tissue reaction of an ion-releasing resin modified glass-ionomer cement with claimed bioactivity (ACTIVA bioactive resin) restorative material versus Mineral Trioxide Aggregate High Plasticity (MTA HP) and bioceramic putty iRoot BP Plus. Fifty-six adult male Wistar rats were assigned into 4 equal groups (14 rats each). A surgical intramedullary bi-lateral tibial bone defects were performed in rats of the control group I (GI) and left without any treatment to be considered as controls (n = 28). The rats of groups II, III and IV were handled as group I except that the tibial bone defects were filled with ACTIVA, MTA HP and iRoot BP, respectively. In all groups, rats were euthanized after one month and specimens were processed to histological investigation, SEM examination and EDX elemental analysis. In addition, semi-quantitative histomorphometric scoring system was conducted for the following parameters; new bone formation, inflammatory response, angiogenesis, granulation tissue, osteoblasts and osteoclasts. The clinical follow-up outcome of this study revealed the recovery of rats after 4 days post-surgical procedure. It was observed that the animal subjects returned to their routine activities, e.g., walking, grooming and eating. The rats showed normal chewing efficiency without any weight loss or postoperative complications. Histologically, the control group sections showed scanty, very thin, new bone trabeculae of immature woven type located mostly at the peripheral part of the tibial bone defects. These defects exhibited greater amount of thick bands of typically organized granulation tissue with central and peripheral orientation. Meanwhile, bone defects of ACTIVA group showed an empty space surrounded by thick, newly formed, immature woven bone trabeculae. Moreover, bone defects of MTA HP group were partially filled with thick newly formed woven bone trabeculae with wide marrow spaces presented centrally and at the periphery with little amount of mature granulation tissue at the central part. The iRoot BP Plus group section exhibited an observable woven bone formation of normal trabecular structures with narrow marrow spaces presented centrally and at the periphery showed lesser amount of well-organized/mature granulation tissue formation. Kruskal Wallis test revealed total significant differences between the control, ACTIVA, MTAHP and iRoot BP Plus groups (p < 0.05). Meanwhile, Mann–Whitney U test showed significant difference between control and ACTIVA groups, Control and MTA HP groups, control and iRoot BP Plus groups. ACTIVA and MTA HP groups, ACTIVA and iRoot BP Plus (p ˂ 0.05) with no significant difference between MTA HP and iRoot BP Plus (p > 0.05). The elemental analysis outcome showed that the lesions of the control group specimens were filled with recently created trabecular bone with limited marrow spaces. EDX tests  indicated a lower degree of mineralization. Lower amounts of Ca and P was expressed in the mapping analysis compared with other test groups. Calcium silicate-based cements induce more bone formation when compared to an ion-releasing resin modified glass-ionomer restoration with claimed bioactivity. Moreover, the bio-inductive properties of the three tested materials are likely the same. Clinical significance: bioactive resin composite can be used as a retrograde filling. Proper retrograde filling should exhibit good bioactivity and biocompatibility. Also, it shows the characteristics of wet setting, easy manipulation, bonding to tooth substrate and radiopacity2.Retrograde filling is indicated for teeth that cannot be treated with routine orthograde root canal therapy and history long-term orthograde RCT failures. Nowadays, several retrograde fillings are well-known such as mineral trioxide aggregate (MTA), zinc oxide eugenol and amalgam. The ideal material should possess certain criteria and thus selecting the most efficacious material is one of the essential prerequisites for obtaining successful treatment outcomes4. Mineral Trioxide Aggregate is one of a calcium silicate-based biomaterial which was introduced in 1993 and since then it has been widely used by endodontists for the treatment of roots’ perforation, apexification as a root-end filling material5. It has been reported that MTA is considered as the gold standard for retrograde filling. This is attributed to its high biocompatibility, osteoinductivity, as well as, excellent chemical and physical properties. Conversely, this category of retro-grade filling suffered from certain limitations e.g. low-compressive strength, high alkalinity and relatively long-setting time6. To overcome the drawbacks of MTA, water-based bioceramic calcium silicate cements have been developed as a fully laboratory-synthesized biomaterials for root canal filling and repair applications, requesting an improvement in their clinical performance7. These materials showed a potent antibacterial activity, sealing ability and good biocompatibility. They are a convenient to utilize and supplied as ready-to-use paste in a white premixed putty form. The bioceramic putty absorbs water and moisture from the surrounding environment and initializes hydration of bi-calcium silicate and tri-calcium silicate to precipitate hydroxyapatite as an end product9.A recently introduced category of an altered ion-releasing RMGI restorative materials has shown a promising results in the field of restorative dentistry10. Also, it has been reported that ProRoot MTA when embedded in the mandibular symphysis of guinea-pigs showed declining toxicity over time with excellent biomineralizing and biological behavior11. A mild-to-moderate chronic inflammatory response with multinucleated and lymphocytes infiltration accompanied with formation of thin fibrous capsule/ dystrophic calcification could be observed one month after implantation of MTA Angelus in rat alveolar sockets, however this inflammatory response was gradually resolved within the following three months12. Likewise, in a rat femur model ProRoot MTA implantation showed better biological performance with lower inflammatory response and increased bone formation over longer follow up periods13. In addition, in a rabbit femur model, ProRoot MTA and an experimental dicalcium silicate cement showed well incorporation with the surrounding bone with diminished inflammatory response14.This category of bioactive cements showed a promising result during grafting of freshly mixed ProRoot MTA implants in mandibular bony cavities of guinea pigs induced minimal inflammation with favorable healing responsehttps://www.nc3rs.org.uk/arrive-guidelines) were followed.Thus, the main purpose of the present study was to assess the intramedullary bone tissue reaction to an ion-releasing resin modified glass-ionomer restoration with claimed bioactivity  versus MTA-HP  and iRoot BP Plus  as two calcium silicate-based cements of different properties in a rat model. This study was conducted to test the null hypotheses that that there were no significant difference between ion-releasing RMGI restoration and the tested 2 calcium silicate-based cements in intramedullary bone reaction and elemental structures. In performing this study, the guidelines of the Animal Research: Reporting In Vivo Experiments and the ARRIVE Checklist  of 0.05, an effect size (f) of 0.40, a power of 0.95 and number of groups was 4. The estimated sample size was 112. (28 rats/ group) and as the surgical defects were performed bilaterally (14 rats/ group); thus, the sample size was 56 rats.16. All experimental steps were performed after obtaining the approval from the ethical committee of Central South University for animal experiments (No: 20180025). The rats were housed at a temperature under 22 °C and at 65%-70% relative humidity. All rats were maintained in a 12-h light and 12-h dark cycle with normal dietary nutrition.Fifty-six adult, male, pathogen-free rats , weighing approximately 180–200 g were obtained from Institute of Basic and Clinical Pharmacology Research Centre of Xiang Ya Medical school, Central South University. Rats were selected and housed in standard cages and used in accordance with the guide for care and use of laboratory animals and the surgical procedures were conformed according to the guidelines of ISO 10993-2  and ISO 10993–1 https://www.randomizer.org/) was used to randomly assign the rats into 4 equal groups , 14 animals per each group. A surgical intramedullary bi-lateral tibial bone defects were performed in rats of the control group I (GI) and left without any treatment to be considered as controls (n = 28). The rats of groups II, III and IV were handled as group I except that the tibial bone defects were filled with with ACTIVA , MTA HP , and iRoot BP Plus , respectively.The Research Randomizer software package . Bilaterally, the fur leg was clipped, and operation site was wrapped in a sterile fashion with 0.12% chlorhexidine and the residual antiseptic was removed with sterile moistened gauze. A one cm longitudinal incision along the anteromedial border of tibia was performed using # 11 scalpel. The medial diaphyseal face of the tibia was exposed via blunt dissection using blunt scissor to expose a full mucoperiosteal flap with care taken during splitting the muscle to avoid injury to the tibial arterial and its venous return. A non-critical sized circular bony defect At of 2 mm diameter throughout the full cortical thickness and reaching the medullary space was performed using surgical round bur fitted on high-speed handpiece with copious irrigation of saline. Bony defects were filled manually with the tested materials according to the manufacture instructions using plastic spatula for the ion-releasing RMGI (ACTIVA) restorative material. Moreover, a stainless steel spatula was used for MTA HP and iRoot BP Plus cements, until extrusion occurred into the medullary space. Then, restorative material was light cured and cements were left to set. The bony defects in the control group were left to heal spontaneously without filling. The overlying muscles and skin were sutured with No. 0/3-resorbable suture.Floxacillin antibiotic of 1 g and ketolac tromethamine analgesic of 10 mg were given for 4 days post surgery with a dose of 0.34 mg/kg. Regular daily checkup for the physical status and the healing course of the rats was performed after surgery. Postoperatively, after one month, each rat was euthanized by administering an anesthetic overdose with ketamine hydrochloride and xylazine hydrochloride  in accordance with ISO standards 10993-1 for prolonged-permanent long-term contact of implant devices for bone tissue. The areas of interest including bone defects were surgically retrieved and prepared for routine histological investigation, SEM examination and EDX elemental analysis. All the surgical procedures were performed by the author A.A and placement of the fillings was blinded in all restorative groups, except, ion-releasing RMGI (which has a unique delivery system).17, technique. Slides were photographed using Leica Microscope  adapted with a DFC420 camera under × 10 magnification to observe the amount of new bone formation, inflammatory cell response, angiogenic process, granulation tissue formation, and then under × 40 magnification to semiquantitatively estimate the number of osteoblast and osteoclast cells. The histological scoring system was performed according to the criteria described by Abid et al.18, with slight modification (Table The tibiae bones (n = 14/group) were fixed in 4% paraformaldehyde, decalcified in 10% EDTA (pH 7.4), longitudinally sectioned at the area of each bone defect. Decalcified tissues were processed in a tissue processor using ascending concentrations of ethanol, embedded in paraffin and sectioned at a thickness of 5 µm. After deparaffinization in xylene and rehydration in graded ethanol, hematoxylin and eosin (H&E) and Masson’s trichrome staining were performed according to Suvarna et al.The retrieved specimens (n = 14/group) were fixed in a solution containing 2.5% glutaraldehyde and 2% paraformaldehyde and then stored for 24 h overnight at 4 °C. The specimens were precisely cut into thin sections using ISOMET 4000, crossing the operation area and passing through the material, to expose the interface with surrounding bone for micromorphological surface analysis. The prepared disks were then washed three times for 15 min with phosphate buffered saline (pH 7.4) and fixed again in a solution of 1% sodium phosphate buffer osmium tetra oxide for 90 min. The exposed specimens’ surfaces were subjected to wet polishing using 600-, 800-, 1200-, 1600-, 1800- and 2000-grit SiC paper, cleaned using ultrasonic cleaner containing deionized water for 30 s. Finally, the specimens underwent graduate dehydration with ethanol. The specimens were dried and coated with gold . Examination of the processed disks is performed using JEOL JSM 6510LV scanning electron microscope .Energy dispersive X-ray analysis (EDX) for elemental microchemical determination was performed randomly for all specimens at the same magnification for the same areas with approximately fixed scanned area to evaluate the relative element content. EDX microanalysis weight was performed on bone material interface in selected frame to analyze entire areas and the elements were calculated from the data to evaluate the degree of mineralization of the newly formed bone. EDX element mapping was performed at the same area to detect the element distribution within the mature and newly formed bone and their presence in the surrounding tissues. Element mapping was performed through the bone-interface-material and were performed to detect the variation content of Ca, P, Si, Ta, Na, K, Al, Ba, Zr, C and O from the mature pre-existing bone towards the implanted material.Generally, recovery of rats was observed after 4 days post-surgery as they returned to their routine activities, such as walking, grooming and routine dietary habits. The rats showed normal chewing efficiency without any weight loss or postoperative complications. In addition, stereomicroscopic pictures for the tibial bone of all groups showed an acceptable healing in the control one, a defect filled with ACTIVA restorative material with better osteointegration with the old bone and an excellent healing was noticed in both MTA HP and iRoot BP Plus groups as they showed marked disappearance of bone defects and the boundaries of osteotomy gaps revealed better osteointegration with the old bone Fig. .Figure 1The outcome of the Kolmogorov–Smirnov normality test showed that data did not follow the normal distribution and accordingly non-parametric statistical analysis was performed. Kruskal Wallis test revealed total significant differences between the control, ACTIVA, MTAHP and iRoot BP Plus groups (p < 0.05). Meanwhile, Mann–Whitney U test showed significant difference between control and ACTIVA groups, Control and MTA HP groups, control and iRoot BP Plus groups. ACTIVA and MTA HP groups, ACTIVA and iRoot BP Plus (p < 0.05) with no significant difference between MTA HP and iRoot BP Plus (p > 0.05).Control group sections showed scanty, very thin, new bone trabeculae of immature woven type located mostly at the peripheral part of the tibial bone defects n = 25) with greater amount of thick bands of typically organized granulation tissue of tightly packed collagen bundles were located centrally and at the periphery (n = 24). The granulation tissue had moderate inflammatory cell response (n = 24)) and profound sprouting angiogenesis (n = 24). Osteoblastic activity was noticed only in the peripheral part of the defect area (n = 20) and less likely at the central one (n = 8) whereas osteoclastic activity was completely absent at both areas  with little amount of mature granulation tissue at the central part (n = 21). The granulation tissue was characterized by mild inflammatory cell response (n = 24), moderate sprouting angiogenesis (n = 26). Osteoblastic (n = 28) and osteoclastic (n = 27) activities were found at the central and peripheral areas . Meanwhile, Mann–Whitney U test showed significant difference between control and ACTIVA groups, Control and MTA HP groups, control and iRoot BP Plus groups. ACTIVA and MTA HP groups, ACTIVA and iRoot BP Plus with no significant difference between MTA HP and iRoot BP Plus  showed an intimate contact and signs of bioactivity with incremental deposition of the newly formed bone along the interface. The representative images showed also a perfect contact with absence of gaps between newly formed bone and the residuals of MTA HP and iRoot BP plus Fig. .Figure 4The EDX results for the control sample representing the normal bone which known to be consists mainly of two compartments (Organic matter) represented by collagen and non-collagenous proteins resulting on the presence of both carbon (kα) and oxygen (kα) lines originally located at 0.2774 and 0.525 keV respectively.The lesions of the control group specimens were filled with recently created trabecular bone with limited marrow spaces. EDX tests  indicated a lower degree of mineralization. Lower amounts of Ca and P was expressed in the mapping analysis corresponded by higher amounts of O and C with wider bone marrow spaces side Fig. A–C. FiguThe histological segment observed by SEM showed the signs of bioactivity along the material-bone interface at the surgical defect with the formation of a layer of newly formed bone when Ion-releasing RMGI (ACTIVA) was put directly in contact with the sample on its medullar side Fig. A–C. EDX The histological segment observed by SEM showed the surgical defect entirely filled with the material and newly created bone filling the medullary space and showed the osteoinductive properties of MTA-HP strictly in contact with the sample on its medullar side. EDX microchemical analysis shows its constituent elements, i.e., Ca, Si, Mg, Al with Bi residues and high amount of P in the samples Fig. A–C. Ca aThe histological segment observed by SEM showed the surgical defect entirely filled with complete integration between material and newly created bone filling the entire medullary space and showed the superior bioactive properties of iRoot BP plus directly in contact with the sample on its medullar side Fig. A–C. Its In the current study calcium silicate cements used for endodontic retrograde filling were tested against newly Ion-releasing RMGI (ACTIVA) restorative material in rat model through intramedullary bone tissue reaction by using H&E histological analysis, SEM morphological and EDX elemental analysis for their bio-functionality and osteo-inductive characteristics in a trial to mimic oral conditions.20. However, some materials were not found to induce any noteworthy interaction with the host living tissues21. These reactions which help the improvement of wound healing and granulation tissue formation. The recent description of biocompatibility is the ability of dental biomaterials to elicit favorable and beneficial reaction with oral tissues during its intended clinical usage22.The old perception of biocompatibility was related to the disappearance of adverse tissue reaction to the tested material23. Therefore, using animal models, like rodents and rabbits are suitable choice for testing biological, regenerative and mineralizing characteristics of new materials in less invasive and non-critical sized surgical procedures24.It has been found that the biocompatibility assessments produced by cell culture assays is not necessarily to be in agreement with animal in vivo biocompatibility implantation test25. It was reported that complete healing occurred following retrograde resin placement with 97% success rate26. Retrograde filling with composite exhibited complete healing in 74% of cases in correlation to 59% of cases treated with amalgam22. These materials showed a higher degree of biocompatibility when subcutaneously implanted in comparison to the calcium silicate-based cements as it decrease the degree of inflammation with a better healing potentiality16.The ingredients of the new ion-releasing resin composite restorative material contain glass particles and a hydrophilic ionic resin matrix that facilitates the diffusion of fluoride, phosphate and calcium ions into the surrounding environmentin-vitro, ex-vivo, and in-vivo laboratory testing are essential to reveal the biological properties of the structure and composition of implanted materials. In-vivo animal models give initial perceptions about the physiological status, biomechanical interaction and the hormonal response against a newly tested materials, starting from rodents and rabbits as small models and ending by dogs and pigs as large ones27. Rabbits and rodent animal models are the key source for studying the biological behavior and regenerative potential in implant studies28. They are excellent candidate for modeling bone infections and extra oral surgical procedures to test the osteoinductive potential of allogeneic and xenogeneic bioactive materials28.A combination of 29.The outcome of this study showed the osteoinductive ability of MTA HP, iRoot BP and ACTIVA with biomineralization and new bone formation around the materials which filled the medullary space of rat tibia. Moreover, inflammation with subsequent necrosis of the cortical bone in contact with the materials was absent. In the analysis process SEM and EDX were adopted as accredited methods for the assessment of the surface morphology, the type and the amount of mineral content of the bone-material interfaceSpecimen analysis showed strong co-relation between calcium and phosphorus (Ca/P) like stoichiometric hydroxyapatite. Zones of mineralization and bone-material interfacial mapping were marked by SEM imaging and EDX mapping; therefore, the differentiation between the old per-existing bone and newly formed bone and cortical and cancellous bone is explained by the differences in minerals constituents expressed by the EDX elemental analysis.The areas of remodeling process and newly formed bone are expressed by the decreased amount of Ca and P ratios in some certain trabecular segments, while, increased amount of Ca and P with the decrease of C, Si and O indicates the directional profile from mineralizing interface towards the old mature bone.30.EDS, EDX, EDAX, or EDXA simply known as Energy-dispersive X-ray spectroscopy representing a direct and powerful tool that enables scientists to analyze and identifying chemical composition of studied samples depending on the core electron ejection with high energy X-ray radiation (Moseley's Law) gives a direct correlation between frequency of emitted radiation and the atomic number of the atomThe data generated by EDX analysis consist of spectra showing peaks corresponding to the elements making up the true composition of the sample being analyzed. In bone, the most frequent application of EDX is measurement of Ca and P content in the extracellular matrix with the calculation of Ca/ P ratio. Local nanomechanical properties have been shown to correlate with Ca levels. Detection of Ca and P along the bone-biomaterial interface confirms the formation of new bone in direct contact with the biomaterial surface. EDX-mapping was performed, because this characterization method is considered an accurate method for detecting elemental distribution among the observation specimen.Histological examination showed that intramedullary mediated bone new formation, osteoblastic differentiation and angiogenic proliferation were implanted by MTA HP, iRoot BP and ACTIVA. Both MTA HP and iRoot BP materials showed strong biocompatibility, as only mild or moderate inflammatory infiltrate was evident quite close to the interface. While ACTIVA showed the least mineralization ability. This might be attributed to its high resinous content which countered its claimed bioactivity.31, and for biomineralization by induction for osteogenic extra-cellular signaling with summoning cascades of cellular differentiation into osteoblasts with osteoid matrix lay down at the surgical site32.The study demonstrated the ability of an Ion-releasing RMGI (ACTIVA) and hydraulic calcium silicate cements to form osteoid matrix with activation of osteoblasts and achieving a direct bond between the materials and the surrounding bone matrix. The formation of favorable environment by calcium silicate cements for osteoblastic differentiation with new bone formation through interfacial active biological ions exchange and its effect on cellular activity could explicate the  response of the tested materials. Bioactivity of a material is related to its chemical reactivity with living tissues through favorable interfacial ion exchange and precipitation reactions33. Therefore, generating a favorable environment at the interface with continuous provision of biologically active ions through the surface for apatite deposition34. In addition, silica Si ions as a byproduct has a role in new bone formation as it encourages the angiogenesis through elevating gene expression which is synergetic with the differentiation of osteoblasts and extracellular matrix synthesis and deposition35.Calcium silicate cements shown in a previous studies to have the ability to provide high alkaline pH in the media and release calcium ions after formation of hydrated silica-rich layer36. These stages including porous colloidal CSH gel and radial acicular CSH crystals, rhombohedral crystals of portlandite, needlelike crystals of ettringite which is a hexacalcium alumina-tetrisulphate hydrate, and calcium mono- sulfoaluminate or calcium mono-carboaluminate. Moisture plays a major role in initiation of apatite formation (bioactivity) and nucleation process of calcium ions on the surface of hydrated calcium silicate particles, it goes through defined stages starting from rapid dissolution of calcium ions after the solid–liquid interface formed on the cement particles forming portlandite. A porous, fine-grained/fibrous and disorganized hydrated silicate gel layer is formed by alkaline etching; containing Si–OH silanol groups and negative surface charges that may act as nucleation sites for apatite formation. Portlandite crystals nucleate inside the hydrating wet paste due to a strong continuous outflux of calcium hydroxide from CSH that occurs during the first hours after mixing. Calcium phosphate and apatite deposit on cement surface as result of exposure to tissue fluids after being placed in vivo37. Calcium hydroxide formed reacts with carbon dioxide in the tissues to form calcified nodules which acted as nucleus for mineralization38. In addition, Zamparini et al.39 revealed that ready-made paste bioceramic materials containing tantalum pentoxide as radiopacifier showed higher calcium and silicate ions release.The setting mechanism of calcium silicate cements starts after hydration by water during the mixing process. The formed calcium silicate hydrate by-products formed after mixing have many phases as reported by Gandolfi et al40. Morrow et al.41 compared the calcium releasing ability of ACTIVA, Dycal and TheraCal in 7 days period, results showed that ACTIVA released an amount of 0.72 μg/mm2 which was less than that released from Dycal (4.88 μg/mm2) and TheraCal 9.12 μg/mm241.The hydrophobic nature of resin component in light-cured calcium silicate containing dental materials make them less soluble than MTA. The pores found in the resin matrix which allow for water penetration with inward outward flux, therefore it could explain their capability of releasing of calcium through solubilization of calcium ions and calcium silicate hydrate and portlandite formation42. It has been also shown that ACTIVA has the ability to form dentinal tubules integrated resin tags with 2.0–2.5 Ca/P ratio across the hybrid layer42.In the context, it has been reported that hydroxy apatite particles formed on the surface of ACTIVA after 30 days of immersion in phosphate buffered saline after utilization of SEM, PulseTor SDD (Silicon Drift Detector) and Energy-dispersive X-ray spectroscopy (EDS) analysis44, and previous study demonstrated the high alkaline pH of iRoot BP and MTA HP and nearly neutral pH for ACTIVA45.Actually, in the present study ACTIVA showed promising biomineralizing response but the tested calcium silicate cements showed better performance in biomineralization, angiogenic activity with neovascularization and formation of capillaries close to the materials. In addition, MTA HP and iRoot BP were incorporated with surrounding bone showing intervening between old and newly formed bone without recognition of the materials exhibiting no interposed connective tissue, resorption lacunae inflammatory response, rejection or necrosis in the adjacent pre-existing bone and medullary side. No osteoclastic activity or multinucleated giant cells observed in the histologic analysis except for a few monocytes and lymphocytes existed. It can be assumed that the high alkaline pH played a significant role in inactivation of osteoclasts as explained in previous studiesIn light of this study outcome, the null hypothesis was rejected. A part of the laboratory work of the study was performed during Covid-19 pandemic time and our research team faced some delay and shifting from the original timeframe due research laboratory closure. Also, further advanced radiographical methods (such as Micro-CT) is recommended in future studies.Calcium silicate-based cements (MTA HP & iRoot BP Plus) and resin-modified glass ionomer restorative material (ACTIVA) exhibit the ability to induce new bone formation through osteo-inductivity. The tested resin-modified glass ionomer restorative material does not induce fibrous encapsulation or any foreign tissue reaction with the direct contact between material superficial layer and mineralized bone matrix.Supplementary Information.