Genetic variation can predispose to disease both through (i) monogenic risk variants that disrupt a physiologic pathway with large effect on disease and (ii) polygenic risk that involves many variants of small effect in different pathways. Few studies have explored the interplay between monogenic and polygenic risk. Here, we study 80,928 individuals to examine whether polygenic background can modify penetrance of disease in tier 1 genomic conditions — familial hypercholesterolemia, hereditary breast and ovarian cancer, and Lynch syndrome. Among carriers of a monogenic risk variant, we estimate substantial gradients in disease risk based on polygenic background — the probability of disease by age 75 years ranged from 17% to 78% for coronary artery disease, 13% to 76% for breast cancer, and 11% to 80% for colon cancer. We propose that accounting for polygenic background is likely to increase accuracy of risk estimation for individuals who inherit a monogenic risk variant. Genetic variation predisposes to disease via monogenic and polygenic risk variants. Here, the authors assess the interplay between these types of variation on disease penetrance in 80,928 individuals. In carriers of monogenic variants, they show that disease risk is a gradient influenced by polygenic background. More recently, polygenic scores have been developed that integrate the effects of many common genetic variants on disease risk5. While the common variants have small individual effects on disease risk, they can cumulatively have large effects—producing, in some individuals, risks equivalent to the strong monogenic variants7. A key question is how monogenic and polygenic risk interact: can disease risk from a monogenic variant that causes major disruption to a specific pathway be meaningfully modified by polygenic risk factors that involve small perturbations to a wide range of cellular pathways?For a range of common heritable diseases, a small subset of the population inherits a rare monogenic variant that causes a large increase in disease risk by disrupting a specific physiological pathwayMYOC variant predisposing to glaucoma, and continuous measures, such as height, body mass index, and cholesterol levels among those with rare monogenic mutations10. With respect to common diseases such as cancer, the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) studied a large number of BRCA1/2 monogenic risk variant carriers recruited from cancer genetics clinics, noting a relationship between a polygenic score comprised of genome-wide significant loci and the risk of breast, ovarian, and prostate cancer12.Recent work has suggested that common variant background modifies the age of disease onset among carriers of high-risk trinucleotide repeats predisposing to Huntington’s disease, the p.Gln368Ter 13. About 1% of asymptomatic adults carry a pathogenic or likely pathogenic variant related to any of these conditions15. Although such variants are associated with several-fold increased risk of disease, it has long been recognized that they have incomplete penetrance and variable expressivity. For example, in one U.S. healthcare system, more than 50% of familial hypercholesterolemia variant carriers and more than 70% of female hereditary breast and ovarian cancer variant carriers remained free of disease well into middle age17. We hypothesized that this incomplete penetrance could be partly explained by polygenic background—a topic that has both scientific implications about disease physiology and clinical implications for genetic counseling.Here, we set out to confirm and extend these prior observations for the three tier 1 genomic conditions highlighted by the U.S. Centers for Disease Control and Prevention—familial hypercholesterolemia, hereditary breast and ovarian cancer, and Lynch syndrome20. Moreover, we compute newer generation polygenic scores for coronary artery disease, breast cancer, and colorectal cancer, which enhance risk prediction over prior scores22.We designed two case-control studies from the UK Biobank and Color Genomics commercial testing laboratory and performed analysis of an independent cohort from the UK Biobank. The two case-control studies increase statistical power by enriching for disease cases, whereas the independent UK Biobank cohort enables assessment of outcomes for both carrier and noncarriers of monogenic risk variants within the context of contemporary medical careLDLR, APOB, and PCSK9—in 6432 coronary artery disease cases and 6420 controls derived from the UK Biobank  cases and 13 (0.20%) controls. The presence of a familial hypercholesterolemia variant conferred a 3.21-fold increased risk of coronary artery disease  when assessed in a logistic regression model adjusted for age, sex, and the first four principal components of ancestry.We first studied the interplay of monogenic risk variants and polygenic scores in coronary artery disease. To identify individuals with monogenic variants causal for familial hypercholesterolemia, we sequenced the three genes related to the condition—nk Table 18. Each 7.We next examined the effect of participants’ polygenic background on risk of coronary artery disease, by computing a previously validated polygenic score in all cases and controlsp-interaction 0.60, Wald Test and Methods).Even among carriers of a familial hypercholesterolemia variant, the observed risk varied substantially according to the polygenic score , intermediate polygenic score (middle three quintiles), or high polygenic score (highest quintile). Compared to non-carriers with intermediate polygenic score, the risk among mutation carriers ranged from 1.30-fold (95% CI 0.39–4.32) for those in the lowest quintile of the polygenic score distribution to 12.61 (95% CI 2.96–53.62) in the highest quintile Fig. . To testWe next examined an independent cohort of 48,812 unrelated UK Biobank participants. A laboratory geneticist identified a familial hypercholesterolemia variant in 130 0.27%) of these participants, 18 (0.87%) cases, and 112 (0.24%) controls, which conferred a 5.08-fold (95% CI 3.01–8.57) increased risk of coronary artery disease at the time of enrollment  cases and 671 (3.9%) controls, corresponding to a 3.48-fold (95% CI 2.81–4.21) increased risk of breast cancer in variant carriers. We then calculated polygenic risk for breast cancer, using a previously validated polygenic score21. As we saw for coronary artery disease, breast cancer risk was strongly affected by polygenic background even for those who carried a pathogenic variant .We next set out to apply the same analysis to breast cancer. We first identified monogenic risk variants by sequencing the Compared to noncarriers with intermediate polygenic score, increased risk among carriers ranged from 2.40-fold (95% CI 1.58–3.65) for those in the lowest quintile of the polygenic score distribution to 6.85-fold (95% CI 4.71–9.96) in the highest quintile Fig. .Fig. 2InBRCA1 and BRCA2 genes  was identified in 76 (0.15%) individuals  of common DNA variants that predispose to coronary artery disease operate via cholesterol-related pathways, with the remainder affecting non-cholesterol-related pathways  and many additional pathways yet to be discoveredBRCA1 or BRCA2 variants are faced with the decision about whether to undergo prophylactic mastectomy as opposed to serial imaging27. At present, up to 50% of women opt for prophylactic mastectomy, but rates are highly variable29. For Lynch syndrome, the current guidelines recommend initiating screening colonoscopy at age 20–25 years and repeating every 1–2 years30. Aspirin has been shown to reduce the risk of colon cancer in Lynch syndrome, but the optimal dose and duration is unknown31. Many experts advocate for prophylactic colectomy in a subset of patients, but this remains controversial32. More accurate risk estimation could better inform the onset and frequency of serial colonoscopies, the risk–benefit ratio for aspirin, or the decision for prophylactic colectomy.Second, our findings indicate that accounting for polygenic background is likely to increase the accuracy of risk estimation for individuals who inherit a monogenic risk variant. An important example is the decision about the timing and intensity of lipid-lowering therapy for individuals with familial hypercholesterolemia. Here, we find a broad spectrum of risk of coronary artery disease in those with a monogenic risk variant across percentiles of the polygenic score that may better inform shared decision making—odds ratios compared to noncarriers ranging from 1.62 to 21.44 and an absolute risk at age 75 years ranging from 17.5% to 77.9%. Refined risk estimates may also help with counseling and clinical decision making for hereditary cancer. In breast cancer, carriers of pathogenic 33, (ii) whole exome sequencing and genotyping array, or (iii) targeted high-coverage sequencing of individual genes and low-coverage sequencing across the genome34. Ongoing efforts to improve the cost and accessibility of these technologies will improve the feasibility of incorporating this information into routine clinical practice.In a clinical setting, assessing both rare monogenic risk variants and polygenic risk requires high-coverage sequencing of genes associated with monogenic risk and an approach to assay common variants across the genome. At present, this can be accomplished by various approaches, including (i) high-coverage whole genome sequencing35. Within any target population, a comparison of predicted and observed disease rates can allow for recalibration of risk estimates, as recently performed for a breast cancer prediction tool36. Second, our analysis focused on the role of monogenic variants in the nine genes associated with the three CDC tier 1 genomic conditions; additional efforts are needed to include variants in other known or newly discovered genes in the future. Representative examples of such genes include those encoding lipoprotein lipase (LPL), where variants increase risk of coronary artery disease via perturbation of triglyceride metabolism, and checkpoint kinase 2 (CHEK2), which increases risk of breast cancer via perturbation of DNA repair pathways38. Third, we aggregated together all pathogenic and likely pathogenic variants for each monogenic condition across the known causal genes. Although this approach is consistent with current clinical treatment guidelines—which recommend similar treatment strategies for each condition regardless of which gene is implicated—risk is known to vary somewhat according to specific gene or variant . Controls included participants free of any self-reported or documented history of coronary artery disease. Only independent samples were used by removing one from a pair of related individuals whenever genetic relationship was closer than second-degree.The study populations consisted of two case-control studies and one national biobank cohort. First, a coronary artery disease case-control study of 12,852 participants (6432 cases and 6420 controls) was derived from the UK Biobank, a prospective national biobank study that enrolled middle-aged adult participants between 2006 and 201020. Since participants derived from commercial genetic testing companies tend to be enriched for relatedness, participants that were related (up to second degree) and participants ascertained through Color Genomics’ cascade screening program were not included in this study.Second, a breast cancer case-control study consisted of 19,264 women  who underwent clinical grade genetic testing for hereditary breast and ovarian cancer syndrome at a commercial testing laboratory . Breast cancer case ascertainment was based on self-report at the time of enrollment19. There was no overlap between this cohort and the 12,852 participants of the case-control study. Extensive clinical data including diagnosis of coronary artery disease and cancer are available on all participants. Coronary artery disease was defined based on self-report of heart attack/myocardial infarction, hospitalization records confirming a diagnosis of acute myocardial infarction or ischemic heart disease, coronary revascularization procedures (coronary artery bypass graft surgery or percutaneous angioplasty/stent placement), or death registry data indicating ischemic heart disease or myocardial infarction as a cause of death. Breast and colorectal cancer were each defined based on self-report of the diagnosis, hospitalization records, cancer registry data specifying type of cancer, and death registry. For each of the three diseases, we considered the earliest date at which the diagnosis was ascertained as the diagnosis date. All participants diagnosed at dates prior to enrollment in the UK Biobank were considered prevalent at baseline, while participants diagnosed after enrollment were considered incident .52. Libraries were constructed and sequenced on an Illumina HiSeq sequencing using 151 bp pair-end reads53. An Illumina Nextera Exome Kit was used for in-solution hybrid selection. Sequencing reads were aligned to the human reference genome build GRCh37.p13 using the Burrows–Wheeler Aligner algorithm54, and aligned non-duplicate reads were locally realigned and base quantiles were recalibrated using the Genome Analysis Toolkit software56. Variants were jointly called using the HaplotypeCaller module of the Genome Analysis Toolkit. We removed samples with contamination >10% (n = 0), samples with <80% of target bases at 20× coverage (n = 0), putative sex chromosome aneuploidy (n = 17), outliers for heterozygozity (n = 4), genotype call rate <95% (n = 6), and samples for which there is no genotyping array data (n = 3). There were also 27 individuals excluded due to sample relatedness. Variants from the remaining 12,852 unrelated samples were carried forward for further analysis. The mean target coverage was 75×, and 91.1% of target bases were captured at >20× sequencing depth.Whole exome sequencing on 12,909 samples from the coronary artery disease case-control study derived from the UK Biobank was performed at the Broad Institute of MIT and Harvard  as described previously20. The Color Genomics laboratory is in compliance with Clinical Laboratory Improvement Amendments (number 05D2081492) and College of American Pathologists (number 8975161). In brief, sequencing reads were aligned to the human reference genome build GRCh37.p12 using the Burrows–Wheeler Aligner algorithm, and duplicated and low-quality reads were discarded. Variants were then jointly called using the HaplotypeCaller module of GATK3.4 and SAMtools version 1.820. A no template control and two positive controls containing a set of known variants were included in every batch of samples. Strict coverage requirements (20 unique reads for each base) were used, and median coverage ranged between 200× and 300×.For the 19,264 samples from the breast cancer case-control cohort, target-enrichment sequencing was performed at the laboratory of Color Genomics  as previously described19, and sequencing reads were aligned to the human reference genome build GRCh38 using the Burrows–Wheeler Aligner algorithm. Coverage exceeded 20× at 94.6% of sites on average. Variant calls through two separate pipelines, an SPB pipeline that used WeCall (GenomicsPLC) and GLnexus software and a functional equivalence (FE) pipeline, were made available by the UK Biobank for 49,960 samples58. We included variants from the FE pipeline that were also present in the SPB pipeline. We excluded 222 samples for which there were no genotyping data available (n = 51) or that failed additional sample quality control using genotyping data: heterozygous missingness outlier (n = 112), putative sex chromosome aneuploidy (n = 56), and discordance between reported and genetic sex (n = 20). There were also 926 individuals excluded due to sample relatedness. The remaining variants on 48,812 unrelated participants were carried forward for further analysis. We converted PLINK-formatted files to VCFs and performed a liftover from GRCh38 to GRCh37.p1359.Finally, whole exome sequencing of 49,960 UK Biobank participants was performed at the Regeneron Genetics Center using 75 base pair paired-end reads with two 10 base pair index reads on the Illumina NovaSeq 6000 platformLDLR, APOB, and PCSK9). We then filtered the observed variants to a candidate list of variants that excludes synonymous variants or variants present at allele frequency of >0.005 in each racial subpopulation of the gnoMAD Genome Aggregation Database, a publicly available population allele frequency database of 141,456 human exomes and genomes47.In the 12,852 samples from the coronary artery disease case-control cohort, the analysis was limited to the protein-coding regions and canonical splice sites of three familial hypercholesterolemia genes , hereditary breast and ovarian cancer syndromes (BRCA1 and BRCA2), and Lynch syndrome . We filtered the observed variants to a candidate list of variants that excludes synonymous variants or variants present at allele frequency of >0.005 in any racial subpopulation of the gnomAD Genome Aggregation Database47. We also performed additional variant quality control filters to exclude variants that fall in low complexity regions, variants that fall in regions with segmental duplications, or variants that did not pass the threshold for the random forest algorithm of gnomAD60. No individual with more than one pathogenic or likely pathogenic variant was identified in any of the three study populations.In the 48,812 participants form the UK Biobank cohort, the analysis was limited to the protein-coding regions and canonical splice sites of nine genes for any of the three genomic conditions: familial hypercholesterolemia -certified clinical geneticist, blinded to the phenotype of the participants, at the Partners HealthCare Laboratory of Molecular Medicine . In summary, the ACMG/AMP criteria for classifying variant pathogenicity look at the effect of the variant on the gene, any previous reports of pathogenicity of the variant, functional studies supporting the damaging effect of the gene, and the prevalence of the variant in cases with the disease and controls23.For the 12,852 coronary artery disease case-control study and the 48,812 UK Biobank cohort, candidate variants were filtered to select variants meeting clinical criteria of pathogenicity (pathogenic or likely pathogenic) based on American College of Medical Genetics and Genomics (ACMG)/Association of Molecular Pathology (AMP) criteria23.Similarly, in the 19,264 breast cancer case-control study from Color Genomics, variant classification was reviewed and signed out by an American Board of Genetics and Genomics (AMBGG)-certified clinical geneticist following criteria for pathogenicity for hereditary breast and ovarian cancer syndromes based on ACMG/AMP criteriaFor each of the three tier 1 genomic conditions, the association of monogenic carrier status by gene with disease was calculated using a logistic regression model with age, sex (except for breast cancer), and the first four principal components of ancestry  using the PLINK2.0 score function52.We used three previously validated polygenic scores for coronary artery disease, breast cancer and colorectal cancer containing 6,630,150, 3820, and 95 variants, respectively~ PC1 + PC2 + PC3 + PC4). We then used the residuals from these models as the ancestry-corrected polygenic score and created reference distributions for each phenotype based on the ancestry-corrected scores . A total of 14,669 variants were removed and the new score consisted of 6,615,481 variants. We then obtained the odds ratio of disease per standard deviation of the modified score in carriers and noncarriers of familial hypercholesterolemia variants, using a logistic regression model adjusted for age, sex, and the first four principal components of ancestry. Second, we sought to remove the impact of the polygenic score on LDL cholesterol by using the residuals from a linear regression model that additionally included a previously derived polygenic score for LDL cholesterol consisting of 2,013,592 variants (PSCAD ~ PSLDL + PC1 + PC2 + PC3 + PC4)61. Similarly for this residualized score, we calculated the odds ratio of coronary artery disease per standard deviation in carriers and noncarriers of rare variants, using a logistic regression model adjusted for age, sex, and the first four principal components of ancestry.We conducted two sensitivity analyses to test the hypothesis that the risk gradient according to the coronary artery disease polygenic score operated via pathways largely unrelated to cholesterol. First, we removed all variants from the coronary artery disease polygenic score that are within 1 megabase of the three familial hypercholesterolemia genes  to a polynomial model with additional higher degree of nonlinear terms (square and cube) of ancestry-corrected polygenic score for each phenotype, separately (Model 2) , the odds ratio for disease was calculated in a logistic regression model with age, sex, and the first four principal components of ancestry as covariates. In the case of breast cancer, only females were included and sex was not a covariate. Non-carriers with intermediate polygenic scores served as the reference group.In the coronary artery disease and breast cancer case-control studies, participants were stratified into three groups according to their polygenic score—low, intermediate, or high defined as the lowest quintile, the middle three quintiles, and the highest quintile of the polygenic score distribution, respectively, as we and others have performed previously11. Formal tests of interaction were performed by including an interaction term—polygenic score × monogenic mutation carrier status—within logistic regressions models. These results should be interpreted within the context of limited statistical power to detect non-additive interaction given the number of individuals carrying monogenic risk variants. To assess this power, we performed a post hoc power calculation using the R package waffect65. Within this framework, we fixed the study size and effect size of the polygenic score in noncarriers to the observed values, and subsequently simulated variable polygenic score effect size in the monogenic variant carriers. For coronary artery disease, given an observed odds ratio per standard deviation increase in the polygenic score in noncarriers of 1.74, we had 80% power to detect a different effect size in carriers if the odds ratio was <0.71 or >5.79 at an alpha of 0.05. In this study, the observed odds ratio per standard deviation of polygenic core in monogenic variant carriers was 2.31, corresponding to a p-value for interaction of 0.60 . For breast cancer, given an observed odds per standard deviation increase in the polygenic score in noncarriers of 1.57, we had 80% power to detect a different effect size in carriers if the odds ratio was <1.25 or >2.01. In this study, the observed odds ratio per standard deviation of polygenic core in monogenic variant carriers was 1.44, corresponding to a p-value for interaction of 0.94 . Within the cohort of 48,812 UK Biobank participants, the p-values for interaction were similarly non-significant—0.07, 0.53, and 0.49  for coronary artery disease, breast cancer, and colorectal cancer, respectively.The data from the coronary artery disease and breast cancer case-control studies suggested roughly additive effects between monogenic risk variants and polygenic risk, consistent with prior reportsWithin the cohort of 48,812 UK Biobank participants, to estimate the odds ratio of disease by monogenic variant status and polygenic score percentile for each of the three diseases, we calculated the odds ratio of prevalent disease using a logistic regression model with enrollment age, sex, and the first four principal components of ancestry as covariates—except for breast cancer analyses, which were restricted to females. The predicted odds ratios were then calculated by referencing the risk of noncarriers with median polygenic score and conditioning on the mean value of each other covariate.67. These models included carrier status, polygenic score, sex, and the first four principal components of ancestry as covariates—except for breast cancer analyses, which were restricted to females. To estimate the probability of disease, we used the monogenic and polygenic effects from the model and standardized the remaining covariates at their mean. The probability of disease by time t was estimated by F(t) = 1−S(t), where S(t) is the survivor function, estimated by the survfit function from the R survival package.A second analysis estimated the probability of disease by age 75 years as a function of monogenic variant carrier status and polygenic score. We fit a Cox proportional hazards model with age as the time-scale, defining the time-to-event as the age at which the diagnosis was first ascertained in cases and the age at the most recent follow-up in controlsp < 0.05, and two-sided p values were used.Statistical analyses were performed using R software, version 3.5 . Statistical significance was set at Further information on research design is available in the Supplementary InformationReporting Summary


Tilapia fish skin has demonstrated promise as a stable and practical biological dressing to be used in wound and burn management. However, the appropriate sterilization technique of the Tilapia fish skin is crucial before its clinical application. The standard sterilization technique must eliminate harmful pathogens but maintain the structural and biochemical properties that could compromise the dressing function. This study investigated and compared the efficiency of three sterilizing agents; chlorhexidine gluconate 4% (CHG), povidone iodine 10% (PVP-I), and silver nanoparticles (25 μg/mL) (AgNPs), at three different times  on Tilapia fish skin based on the microbial count, histological and collagen properties. Among the sterilization procedures, AgNPs showed rapid and complete antimicrobial activity, with a 100% reduction in microbial growth of the fish skin throughout the treated times. Furthermore, AgNPs did not impair the cellular structure or collagen fibers content of the fish skin. However, CHG and PVP-I caused alterations in the collagen content. This study demonstrated that the AgNPs treatment of Tilapia fish skin provided sterile skin while preserving the histological properties and structural integrity. These findings provide an efficient and quick sterilization method suitable for Tilapia fish skin that could be adopted as a biological dressing. Biological dressings, such as amnion, allografts, xenografts, bioengineered tissues, and cultured-wound healing cells, have been used recently for topical treatment of burns and skin wounds . CollageOreochromis niloticus) skin as a potential natural material in the management of burns and wounds arose because of its collagenous, histological, and mechanical comparability to human skin  fish , obtained from The Aquatic Medicine Unit's tanks, Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt. The fish were euthanized physically by decapitation according to The American Association of Zoo Veterinarians (AAZV) Guidelines for euthanasia  , povidone iodine 10% (PVP-I) , and silver nanoparticles (AgNPs) (25 μg/mL). The Tilapia skin strips were allocated randomly into four main groups (3 skin strips for each); CHG, PVP-I, and AgNPs groups. Each group was then incubated for different contact time points . In addition to a control group (C) treated with normal saline for the three different times. The tilapia skin strips were subjected to bacteriological and histological evaluation following each sterilization treatment at the indicated time points.3) crystal  was added and stirred well. This mixture was put into a dark glass bottle and autoclaved. The resulting solution was clear yellow in color indicating the silver nanoparticles formation.Stable silver nanoparticles (AgNPs) <100 nm were synthesized in a typical one-step protocol as described before . BrieflyThe stock solution of AgNPs was kept in dark glass bottles away from direct sunlight and at room temperature (25°C). The concentration and size of the particles were measured before using. The morphology and size of AgNPs were measured by transmission electron microscopy (TEM) (JEOL-JEM-100CX II) at the Electron Microscopy Unit, Assiut University . The con2 was aseptically swabbed for microbial count. Each of Plate count agar “HMESIA- Ref -M091A,” Manitol Salt Agar “Oxoid CM0085,” MacConkey Agar OMRI W/O Crystal violet “Biolife “and DifcoTM Potato Dextrose Agar- Ref 213400 were used for Total viable bacteria, Staphylococci, Coliforms, Yeasts and Molds count, respectively. The Pour plate method was used for the enumeration of colony-forming units (CFU/cm2) on Plate Count Agar. The swab was transferred immediately into 10 ml sterile nutrient broth. One milliliter from the broth was transferred aseptically into a sterilized petri-dish in triplicate. Ten to fifteen milliliter of the dissolved Plate count agar media was cooled in water bath to a temperature of 44–46°C and poured aseptically into each plate. The dish was moved in a circular motion several times to ensure homogeneity of the sample with the medium. The plate was left until the hardening of the medium, and then incubated upside down in the incubator at a temperature of 37°C for 24 h. After incubation period the total number of each replicate of the sample was counted and the mean extracted and multiplied by the inverse of dilution. The resulting colonies expressed as CFU/ml. The same technique was used for Staphylococci, Coliforms, Yeasts and Molds count. Staphylococcal count was plated on Manitol Salt Agar “Oxoid CM0085” at 37°C for 24 h, Coliform count on MacConkey Agar OMRI W/O Crystal violet “Biolife at 37°C for 18–24 h, and Yeasts and Molds was plated on DifcoTM Potato Dextrose Agar- Ref 213400 incubated at 28 ± 2°C for 24–72 h.Microbiological evaluation of the fish skin strips was performed according to APHA  and QaziThe efficiency of each sterilization procedure was calculated by comparing the bacterial count before and after each treatment for each contact time via the following equation:0 – C)/C0 × 100, where (C0) is the initial bacterial count (control negative) and (C) is the count of bacteria after a certain contact time of the sterilizing agent (Disinfection efficacy % = (Cng agent .Specimens (0.5 × 0.5 cm) of the Tilapia skin strips were collected from the groups following each sterilization treatment at different times and fixed in 10% neutral buffered formalin. The formalin-fixed skin samples were routinely processed and embedded in paraffin. They were then sectioned at 5 μm thickness and stained with Mayer's hematoxylin  and eosin . Afterwards, the slides were examined microscopically and the histological evaluation was performed in a blind fashion on coded samples, and a comparison was made with the sections from the control-treated group. Histological evaluation to assess integrity and organization of collagen fibers was based on 0–3 scale , 36. ColSirius red stain was used for evaluation of the collagen content . ParaffiUsing threshold area fraction determination, the percentage of collagen positive area was calculated using ImageJ . The amoAn improved computing technology has been used to process color images by segmenting background object pixels from the foreground object , 39, makP-values of < 0.05 were considered statistically significant. Figures were generated using Adobe Photoshop CS6 and Prism 5 version 5.03.Statistical analyses were performed by one-way ANOVA using GraphPad Prism software version 5.03 . The effectiveness of chlorhexidine gluconate 4%, povidone iodine 10%, and silver nanoparticles (25 μg/mL) on the microbial count of the fish skin at the different three contact time points was summarized in Histological analysis of the fish skin samples from the control group showed compactly arranged bundles of collagen fibers in the dermis and occasionally focal epithelial coating and superficial melanophores were seen. The collagen fibers were well organized and distributed in a parallel pattern with no evidence of disaggregation .P < 0.0001).Fish skin treated with CHG for 5 min showed well organized and parallel collagen fibers  with milP < 0.0001).In the PVP-I-treated group, the structure of the collagen fibers has not been changed after 5 min treatment . DisorgaHowever, there was no change in the organization pattern of disaggregation of the collagen fibers in the AgNPs-treated group after 5–15 min treatment in comparing with control samples .P < 0.0001) after sterilization for 15 min with CHG or PVP-I  compared to CHG or PVP-I. Furthermore, the rapid antimicrobial activity of AgNPs is an obvious option so that the operative time is not extended beyond the minimum necessary.By controlling the bioburden, AgNPs facilitate less toxic metabolites production, which result in a better collagen fibers preservation in the Tilapia skin. Furthermore, eukaryotic cells are less prone to silver toxicity than prokaryotes, which is thought due to an increased degree of structural and functional stability in eukaryotes . HoweverSterilization of Tilapia fish skin, used as a biological dressing, using AgNPs is effective with a 100% reduction against microbial growth and without any change in the collagen content at three different times . This is the first study to report the excellent performance of AgNPs as a quick and efficient sterilizer procedure for the Tilapia fish skin, relevant for clinical use as a biological dressing.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by National Ethical Committee of the Faculty of Veterinary Medicine, Assiut University, Assiut, Egypt.AI, SK, and MS designed the study, helped data interpretation, discussed the results, and edited the manuscript. AI, SK, NK, and MS performed the experiments. AI, SK, DH, and MS analyzed the data. MS wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the name of development, industries discharge their wastewater, which contains different Metallic species and massive organic load into the next-door river system. In this study,we assess the impact of organic and inorganic contaminations on Hasdeo River at Korba region, which is fifth critically polluted city in India. Hear, a new approach for water quality indexinglike Water quality index (WQI), Heavy metal pollution index (HPI) and metal index (MI) has been proposed to represent pollution due to heavy metals in river system. The sample's pollutionparameters and heavy metals contamination is exceed from BIS or WHO standards of drinking water . WQI shows that the entire water samples are not suitable for drinking andaquatic life but they are safe only for irrigation. HPI and MI calculation revels that more than 95% sampling sites are critically polluted with heavy metals. Thus, a high level of industrializationdeterioration of river water quality is recorded for adequate action. Rivers are the most important fresh water resource in the world and supply water for many purposes. India has been blessed huge amount of surface water in the form of rivers. In thename of development, Industries discharge their treated or partially treated wastewater, which contains different Metallic species and massive organic load into the next-door river systems,2. Not oThe Hasdeo River, which is more than 100 km drainage system in human province, India, has been receiving metals along with organic matter from mining, coal, paper industries for last15 years in korba region, Chhattisgadh, India. Korba is highly industrialized and important area that contributes nationally and globally to the economy of India. As a consequence of thisindustrialization, CPCB  identifiWater quality index (WQI), Heavy metal pollution index (HPI) and metal index (MI) are the mathematical technique used to transform large quantities of water quality parameters intoa single number, which provides a simple and understandable tool to interpret quality and possible uses of a water body like drinking, irrigation, fishing. Keeping above problems in ourmind the objectives of this study were: 1: assessment of current chemical texture of Hasdeo River water in terms of organic and inorganic load at different location; and 2: Evaluation ofwater quality in terms of WQI, HPI and MI.The Hasdeo River, a tributary of Mahanadi River originates from the valley of chota Nagpur, hill region of a Deogarh, Chattishgarh, India and flows through Korba, Janjgir-Champa districtand joined in Mahanadi River. The climate in the Korba region is tropical with average temperature 26.6°C and 1420 mm average rainfall. Korba is situated at 22.3595°N, 82.7501°Eon the banks of the Confluence of river Hasdeo.Samples were collected from six different stations, 2 from Hasdeo and 4 from Kesla River in Korba . Samplin-3), chloride (Cl-) and fluoride(F-) were analyzed. Analysis of sodium and potassium was performed by using flame photometer . Sulphate (SO4-) and phosphate (PO4-) was measured byusing double beam UV–Visible spectrophotometer (Perkin Elmer Lambda 35) from turbidimetric and stannous chloride method respectively. Total solid (TS), Total Dissolved Solid (TDS) and TotalSuspended Solid (TSS) were measured using gravimetric method. Total Hardness (T- hard as CaCO3) was determined by the EDTA titrimetric method. Acid–base titration was used to determinetotal carbonate and bicarbonate alkalinities. Color was measured through visual comparison method, Chemical Oxygen Demand (COD) through open reflux method and Biological Oxygen Demand(BOD) through 5-day method. Heavy metals: chromium, cadmium, copper, iron, nickel, lead, zinc and manganese were acid digested with nitric/ perchloric acid mixture (5:1) and measuredby using Inductively coupled plasma spectrophotometer (ICP) . All observations were recorded in triplicate and their average valuesare reported.All the physicochemical parameters of river water were analyzed by using the standard protocols of APHA . During WQI has been calculated from the weighted arithmetic index method in the following steps ,11:Equation →1 (see PDF) Equation →2 (see PDF)Where, Vi = measured value of ith water quality parameter present, Vo = ideal value of ith water quality parameter in pure water, Vo = zero for all parameters except for pH =7.0 andDO = 14.6 mg/l . Si = stUnit weight (Wi) for various water quality parameters is inversely proportional to the recommended standards for the corresponding water quality parameters.Equation →3 (see PDF)Where K is the proportionality constant of the "Weights" for various water quality parameters Equation →4 (see PDF)The water quality has been classified on the basis of WQI into 5 Classes: WQI 0–25 excellent, grade A; WQI 26–50 good, grade B; WQI 51–75 poor, grade C; WQI 76–100 very poor grade Dand WQI >100 unfit, grade E.To determine heavy metal contamination in Hasdeo River water two different quality indices are used in this study. Heavy metal pollution index (HPI) is a powerful technique for theassessment of overall water quality with respect to heavy metals . HPI is Equation →5 (see PDF)Where, Qi = sub index of ith water quality parameter, Wi = unit weightage of ith parameter, n = number of parameters. Wi of ith parameter is defined as inversely proportional to thestandard permissible value (Si) for each parameter ,15.Calculation of Sub index Qi parameter is given by Equation →6 (see PDF)Where, Mi = measured value of heavy metal of ith parameter present, Ii = ideal value or highest desirable value of ith parameter, Si = standard permissible value of ith parameter, thesign (-) indicates numerical difference of the two values, ignoring the algebraic sign. The critical pollution index value for drinking water is 100.The metal index (MI) calculates the relative contamination of different heavy metals separately and manifests the summation of generated components as a representative to deterEquation →7 (see PDF)Where MI = metal index, Ci = concentration of each element, MAC = maximum allowed concentration for each element, subscript i = ith sample. The higher value of MI affects water qualitymore and is more harmful for human health. MI value >1 is concluded as threshold of warning . MI is cOne-way analysis of variance (ANOVA) and Tukey's multiple comparison tests were used to compare the mean values of the different physico-chemical parameters for all the sampling sitesand to identify the homogeneous type of the data sets. Pearson correlation matrix was also calculated by the Pearson correlations test for the different physicochemical parameters and heavymetal concentrations of river water from different sampling sites. Statistical analysis was carried out using the statistical package for the social science, version 22 . P < 0.05 was considered as statistically significant.The physicochemical parameters of river water were analyzed statistically and results are given in Temperature is an essential and changeable environmental factor that affects overall quality of water. During daytime the mean temperature recorded at all sampling sites were in therange of 24.7-25.5 °C. Temperature was almost equal at sampling site K1, K0, K2 and K3 and not showing statistically significant difference .pH of all the sampling sites were 7.4 to 8.0, within a range appropriate limits of Bureau of Indian standard  for wateThe color of river water was significantly different in sampling sites K2, HK1 and HK2 , highest color 112.4±3.33 CoPt  was observed at site K2 might be due to higher accumulation of different organic and inorganic compounds coming from BALCO industry and CSEB fly ash dykes.In this study, almost all the samples had high electrical activity and statistically significant difference among each other  and out of them site HK2 had highestEC value 1815±27 µS/cm which exceeds the permissible limit  of BIS, 2005 might be due to urban discharges.In a liquid, TDS is defined as a measure of the combined content of all substances i.e. inorganic and organic originating from natural sources, urban discharges, industrial waste waterand chemicals used in the water treatment process. The TDS of river water was maximum 1200 mg/L and minimum 202 mg/L at sampling sites HK2 and K1, respectively. These results indicated thatwater from HK2 was containing approximately 6 fold higher TDS than K1, that might be due to the mixing of pollutants through industrial and domestic activities. TDS from K1, K0, K2 and K3were within a range of WHO water quality standard that is 500 mg/L and HK1 and HK2 were exceeded through this range. If this water will be used for drinking purposes it may induces an unfavourablephysiological reaction in the transient consumer and gastro-intestinal infections [Dissolved oxygen (DO) is an important parameter to access quality of river water . Its defHigh BOD adversely affects the river water quality and biodiversity. In this study, BOD ranged from 7 mg/L (minimum at K1) to 44.4 mg/L (maximum at HK2), which were above the CPCB standards . ComparaThe metal analysis of different sites is shown in Calculations and result of HPI for all the sampling sites with unit weightage (Wi) and standard permissible value (Si) is shown in Metal index calculations and results are shown in Correlation analysis for 20 physico-chemical parameters and 8 heavy metals from different sampling sites of Hasdeo River were performed .CorrelaWater quality assessment of the river using WQI, HPI and MI calculations shows that the water of Hasdeo and their tributary river is suitable for irrigation purpose but not for drinkingas well as aquatic life.


Background: Necessity of evaluating heat stress in the workplace, require validation of indicesand selection optimal index. The present study aimed to assess the precision and validity ofsome heat stress indices and select the optimum index for using in heavy work activities in hotand dry climates.Methods: It carried out on 184 workers from 40 brick kilns workshops in the city of Qom, centralIran (as representative hot and dry climates). After reviewing the working process and evaluationthe activity of workers and the type of work, environmental and physiological parametersaccording to standards recommended by International Organization for Standardization (ISO)including ISO 7243 and ISO 9886 were measured and indices were calculated.Results: Workers engaged in indoor kiln experienced the highest values of natural wettemperature, dry temperature, globe temperature and relative humidity among studied sections(P<0.05). Indoor workplaces had the higher levels of all environmental parameters than outdoors(P=0.0001), except for air velocity. The wet-bulb globe temperature (WBGT), predicted heatstrain (PHS) and heat stress index (HSI) indices had the highest correlation with thephysiological parameters. Relationship between WBGT index and carotid artery temperature(r=0.49), skin temperature (r=0.319), and oral temperature (r=0.203) was statistically significant(P=0.006).Conclusions: Since WBGT index, as the most applicable index for evaluating heat stress inworkplaces is approved by ISO, and due to the positive features of WBGT such as ease ofmeasurement and calculation, and with respect to some limitation in application of HSI; WBGTcan be introduced as the most valid empirical index of heat stress in the brick workshops. Some of these disorders include skin rash, extreme tiredness, loss of consciousness, muscle cramps, heart - cardiovascular problems and increase in work-related accidents5. In some cases, heat stress can cause the death of workers6.Working in hot processes, especially in hot and dry climates causes increase deep body temperature from normal range because of the imbalance in body temperature and disorders occur due to heat exposure on workers7. Eighty percent of heatstroke can lead to death8. Workers in various occupations such as foundry, construction, and bakery, agriculture and road construction, especially in the hot season prone to heat stress9.Heat stress can lead to symptoms such as headache, syncope, heat exhaustion and in severe cases neurological disorders and heatstroke12. There is not integrated index that be acceptable in variety of different weather13, for this reason, we require to validate the thermal stress indices in order to have a better describe of environmental conditions. Currently, there are several indices to describe the thermal stress in work environments that each of them has advantages and disadvantages 14.Nowadays, on the issues of heat stress management attention has been paid to identify and control of risk factors of heat stress. Therefore, the assessment of thermal stress in the workplace is very important. These assessments can be done using various indices. Most heat stress indices have been obtained through laboratory studies, while the actual conditions in industry are along with vast changes in environmental and physiological variables15. The body's response to heat stress is known as a strain that can be measure by using physiological parameters16 such as body depth temperature  and by correlating them with indices can validate various indices and choose the appropriate and efficient index18.Optimum index should be accurate, useful and applicable for a range of environmental and metabolic conditions, and evaluate workers' exposure without interfering with its performanceIn this study, Qom City, central Iran due to geographical location as hot and dry climate and also because of the large number of brick kilns workshops (563 workshops) and its activity were selected as representative occupational groups that have heavy activity. Heat received by the workforce in this occupation, with exposure to heat, especially in summer, along with heavy activity is natural 19. For this reason and the above-mentioned reasons, in this study the number of heat stress indices (wet-bulb globe temperature (WBGT), heat stress index (HSI), predicted heat strain (PHS), effective temperature (ET), corrected effective temperature (CET) and discomfort index (DI)) were assessed and validated, and finally, an index determined and introduced as a valid index.12.This descriptive and analytical study was performed on 184 men at workshops of brick kilns in the city of Qom in the spring and summer of 2013. A short description of the brickmaking process includes tempering (adding water to the clay soil), molding (putting the clay mix into a mold), drying , firing (laying out and heating the bricks in kiln)20, work metabolism of workers was classified in heavy job groups. To measuring the dry temperature, globe temperature, wet natural temperature and relative humidity we used calibrated WBGT meter . Air velocity was measured by thermal anemometer . These measurements were carried out, according to the standard ISO 724321, in three areas, namely the height of the head, abdomen and ankle height and at three intervals including 8-10, 10-13 and 16-19during work shift5.According to the standard of ACGIH22.In addition, measurement of the physiological parameters was done at the same time of measuring environmental parameters. Skin temperature was measured by skin thermometer device . Ear Carotid artery temperature measured by thermometer , heart (pulse) rate and systolic and diastolic blood pressure measured by of wrist barometer , and oral temperature were measured by oral thermometer simultaneously with measuring environmental parameters in accordance with ISO9886 standardIndices were studied by the following methods:5.To calculate WBGT index in outdoor and indoor environment used equations 1 and 2, respectively WBGT out = 0.7 Tnw + 0.2 Tg + 0.1 Ta Eq.1WBGT in = 0.7 Tnw + 0.3 Tg Eq.2Tnw: Natural wet temperatureTg: globe temperatureTa: dry temperature21.In workplace where workers receive heat by process, in addition to environment, assuming that environment is heterogeneous, calculations were performed by Equation 3 according to standard ISO 7243  head+ (2 × WBGT abdomen) + WBGT feet) /4 Eq.3WBGT=  is equal to:R: energy exchanged by radiation and its value  Eq.7R=4.4 (35-MRTw/m2) is equal to:C: energy exchanged by convection and its value (0.6 (35-ta) Eq.8C=4.6VEmax: maximum energy excreted from the body by evaporation in the working environment:0.6 (56-Pa) (w/m2) Eq.9Emax=7VPa: pressure of water vapor in air (mb)m/s)V: Air flow rate (Predicted heat strain) PHS (index):24 is used to calculate PHS (predicted heat strain).The software recommended in standard ISO793325. As standard, the TWA (Time Weighting Average) index is calculated for different time using the followingequation17:These indices were derived from the specific diagrams and using of some factors including drying temperature, radiation temperature and wet temperature  TWA = (IndexTWA1×T1) + (IndexTWA2×T2) +…. + (IndexTWAn ×Tn)/T1+T2+…Tn  Eq.10IndexFor statistical analysis used Microsoft Office Excel 2010 and SPSS 18 software.The existing tasks at the brick kilns workshop were divided into 4 groups.Those who work on conveyor as conveyors worker.Those who carrying the load .Those who involved in tempering and molding.Those who involved in picking in and picked up brick from the kiln as indoor and outdoor kiln workers.2.Number of workers studied in conveyors, handling, tempering and molding and kiln parts are 41 (22.3%), 55 (29.9%), 22 (11.9%) and 66 (35.9%), respectively. Age of workers was between 9 and 70 years and theirs work experience was between 1 to 37 years. According to heat stress criteria recommended by American conference of governmental industrial hygienists (ACGIH) and the metabolic rate of all participants (≈415 w), their work was assigned heavyP<0.05; one-way ANOVA).As can be seen in P=0.0001), except for air velocity. Data related to the physiological responses of the workers shown that mean of oral temperature, skin temperature and ear carotid artery temperature in workers of indoor kiln is higher than ones in other sections (P<0.05).The measurement results of environmental and physiological parameters in various sections are presented in P=0.001).In addition, P<0.001). The highest correlation coefficient was found between WBGT and DI .The correlation coefficient between heat stress indices and physiological parameters as well as between various indices with each other is shown in Heat stress on workers in indoor and outdoor environments is the main work-related risk agents, especially in heavy activity and in hot and dry areas such as the city of Qom. Nowadays, various indices are used to evaluate the heat stress that each of them has advantages and disadvantages. Determine optimal index for each workplace, can help to better assessment of environment in terms of thermal stress and therefore be effective to improve health plan.17. This could be due to lack of engineering and management systems in control of heat stress in understudy workshops. In that study17, workers in indoor environments did not receive heat through the process.Results of this study show that all heat stress indices in the indoor environment are more than ones in outdoor that was not consistent with another study13. However, in a steel industry, the indices of HSI, WBGT, ET and CET did not have a significant relationship with deep temperature and oral temperature10. Since these studies are field studies, not controlled and due to difference of thermal stress in various work environments, as well as the possible exposure of workers to other stresses, we cannot make definitive judgment on compliance or non-compliance of these studies.According to 13. Many factors can affect blood pressure such as nutrition, exercise, medication and lifestyle that in this study, due to lack of control of these risk factors, blood pressure changes (including significant or non-significant changes) and cannot be attributed to weather conditions workplace, but certainly these factors have had an intervening role and perhaps be the reason of differences in the results.According to 10. Clantari et al. performed a study in the steel industry, chose optimum index using the correlation between environmental factors and physiological parameters, and introduced P4SR index as the most reliable index for steel industry 10. In addition, Golbabaei et al. with study of comparison between the thermal stress indices and choice of optimal index compared thermal stress indices using correlation between the index and heart rate and select WBGT index as optimum index for hot and humid environment13. Falahati et al. examined the validity of WBGT and P4SR indices using deep temperature, and concluded that the WBGT index was more reliable than the P4SR index17. Chen et al. investigated thermal stress in steel factory workers, and assessed indices with physiological parameters and showed that skin temperature had the highest correlation with WBGT index, besides SWreq index had the greatest correlation with deep body temperature26. Moran et al.27and Frank et al.28used heart rate and deep body temperature to evaluate heat stress.To validate the indices, the considered index should have a strong meaningful relationship with physiological parameters to have the required validityIn this study, optimum index was chosen by studying the correlation coefficient between the various indices with each other as well as with physiological parameters. However, this study showed that the WBGT and heat stress index (HSI) indices had the highest correlation with other physiological parameters among the other heat stress indices; due to the positive features of WBGT such as ease of measurement and calculation, WBGT can be introduced as the most reliable empirical index for assessing the heat stress in heavy activity. WBGT index, as the most applicable index for evaluating heat stress in workplaces for indoor and outdoor environment has been approved by International Organization for Standardization.29, values of this index is too exaggerated.This index has shown a good correlation with environmental factors and physiological parameters 2, 3, and 11. On the other hand, HSI index has relatively high correlation with the physiological parameters and other indices, but according the study of the Di Corleto30. Therefore, the HSI index can be used under certain circumstances and as a supplement index of WBGT.When the airflow rate is equal to zero or close to zero, HSI index estimates more than real; In this case, the results have little value and cannot be evaluatedPHS index is not a good index for brick burning workplace, because this index is suitable for environments where environmental parameters remain constant and not fluctuate and it is applicable for very detailed assessment. According to the results, the environmental parameters are variable in brick kilns workshops and hence to assess heat stress in this job with heavy activity, use of PHS index is not appropriate.There is a suitable correlation between physiological measured parameters and WBGT index, and also, since WBGT index, as the most applicable index for evaluating heat stress in workplaces is approved by ISO, and due to the positive features of WBGT such as ease of measurement and calculation, and also with respect to some limitation in application of HSI; WBGT can be a valid empirical index of heat stress in heavy activities such as brick workshops.This article is part of a research project Supported by Tehran University of Medical Sciences (Grant no: 22877).All authors declare that there are no conflicts of interest.Indoor workplaces (indoor kilns) had the higher levels of most environmental parameters than outdoors. Wet-bulb globe temperature (WBGT), predicted heat strain (PHS) and heat stress index (HSI) indices had the highest correlation with the physiological parameters.  The highest correlation coefficient was found between WBGT and DI (discomfort index(.  WBGT can be a valid empirical index of heat stress in heavy activities such as brick workshops.


We herein report on a male with primary accessory breast cancer in an axilla. A 75-year-old man first noticed a subcutaneous nodule about 2 cm in diameter in the area of his right axilla. The patient underwent extirpation of the mass in a public hospital. Histological examination revealed invasive breast carcinoma of no special type associated with mucinous carcinoma, invasive micropapillary carcinoma and intraductal components. Immunohistochemical analysis showed that the tumor cells were positive for Gross cystic disease fluid protein (GCDFP)-15, mammaglobin and GATA3. Staining for estrogen receptor (ER) and progesterone receptor (PR) was positive, and human epidermal growth factor receptor 2 (HER2) was negative. The Ki67 labeling index (LI) was 33.6%. Imaging revealed no evidence of a primary tumor in any other organ or in the bilateral mammary gland. We performed radical resection of the right axilla, including the scar, and axillary lymph node dissection. The final pathological examination of the surgical specimen showed normal mammary gland tissue that was not connected to the proper mammary gland, and no residual cancer or metastatic lymph nodes. Based on our clinical and pathological findings, this tumor was diagnosed as breast cancer originating from the accessory mammary gland in the right axilla. After surgery, tamoxifen was administered as adjuvant therapy. Since the surgery, 2 years ago, there has been no evidence of recurrence. Hereditary Breast and Ovarian Cancer syndrome was suspected in this case because the patient was a male with breast cancer, and he had two first-degree relatives with breast cancer. This patient had no BRCA mutations on genetic testing. Nonetheless, in cases of male breast cancer, it is necessary to obtain genetic information due to the possibility of hereditary breast cancer, including cancers associated with BRCA gene mutation. Breast carcinoma in males accounts for just 0.6% of all malignant breast neoplasm cases in Japan . The totA 75-year-old man first noticed a subcutaneous nodule about 2 cm in diameter in the area of the right axilla and consulted a public hospital. He was palpated and given a probable diagnosis of a benign tumor. The patient then underwent extirpation of the mass under local anesthesia. The pathological diagnosis of the resected sample was primary breast cancer of the axillary accessory mammary gland. The patient was then referred to our hospital.At the initial visit to our hospital, physical examination showed only a surgical scar with no mass near the right axillary area. A scar was present within the axilla but outside the milk line Fig. . The bilThe excised tumor was 13 × 11 mm in diameter. There was no involvement of skin or skeletal muscle. Histological assessment revealed invasive breast carcinoma of no special type associated with mucinous and invasive micropapillary carcinoma. The mucinous and invasive micropapillary carcinoma components occupied 20% and 10% of the tumor area, respectively. The majority of the carcinoma area was invasive breast carcinoma, though a few intraductal components were also present, as confirmed by immunostaining for p65, CD10 and αSMA. Immunostaining for D2–40 and CD31 and elastica-Masson staining revealed infiltration of fat tissue, without vascular or lymphatic invasion. Immunohistochemical analysis showed that the tumor cells were positive for E-cadherin, Gross cystic disease fluid protein (GCDFP)-15, mammaglobin and GATA3, and were negative for synaptophysin. Staining for estrogen receptor (ER) and progesterone receptor (PR) was positive in 98% of the tumor cell nuclei, and human epidermal growth factor receptor 2 (HER2) was negative in the tumor cells. Ki-67 labeling index (LI) was 33.6% .Computed tomography, magnetic resonance imaging and ultrasonography showed the following: (1) slight enhancement that appeared to be changed after the tumor resection, with no significant local residual tumor Fig. ; (2) no Based on the clinical and pathological findings, this tumor was diagnosed as breast cancer originating from an accessory mammary gland in the right axilla, rather than a cutaneous adnexal carcinoma.We performed radical resection of the right axilla, including the scar from the excisional biopsy, and axillary lymph node dissection under general anesthesia.The final pathological examination of a surgical specimen showed normal mammary gland tissue that was not connected to the proper mammary gland and postoperative fibrotic scarring. No residual cancer or metastatic lymph nodes were found Fig. .Fig. 5ThPostoperative clinical course: the patient recovered uneventfully and was discharged 10 days postoperatively. Tamoxifen at 20 mg/day was administered as adjuvant therapy. The patient has presented no metastatic lesions in the 2 years since the operation and has been receiving hormone therapy. Because there was the possibility that Hereditary Breast and Ovarian Cancer syndrome (HBOC) was the cause of this case of male breast cancer, given that the patient has two first-degree relatives with breast cancer, the patient received genetic counseling after his surgery. Initially, the patient declined genetic testing, but 2 years after his operation, he requested it. However, no clinically significant BRCA1/2 gene mutation was identified.Accessory breast carcinoma in males is rare –3. In adIt is often difficult to distinguish between breast cancer and cancer derived from sweat glands or sebaceous glands, and the presence of carcinoma in situ or ER/PR positivity is used for differentiation . AdditioAccording to data compiled by Maki et al., local radical resection and axillary lymph node dissection is the most common surgical approach, accounting for 64.0% of surgical treatments [In male breast cancer, adjuvant chemotherapy is recommended adheres to guidelines used for women and Tamoxifen is recommended for adjuvant endocrine therapy . In our Guidelines such as the “NCCN Guideline Genetic/Familial High-Risk Assessment: Breast and Ovarian 2019 ver3 ” point oIn addition to BRCA 1/2, associations between breast cancer risk and protein-truncating variants of PALB2, CHEK2, ATM, TP53, PTEN, NF1, Double carrier, CDH1 and NBN have been established . TherefoPhysicians and patients lack awareness of male accessory breast cancer because it is rare. Tumors that develop on and around the milk line should always be carefully examined, given the potential for accessory breast cancer. Additionally, in cases of male breast cancer, it is necessary to obtain genetic information due to the possibility of hereditary breast cancer, including cancers associated with BRCA gene mutation.


We recently reported that bioengineered corneas can be fabricated in vivo usefulness by implantation in an animal model.Here, we generated a partial thickness cornea model based on plastic compression nanostructured fibrin-agarose biomaterials with cornea epithelial cells on top, as an orthotypical model (HOC), or with HWJSC, as a heterotypical model (HHC), and determined their potential in vitro HOC and HHC had moderate levels of stromal and epithelial cell marker expression, whereas in vivo grafted corneas were more similar to control corneas.No major side effects were seen 3 and 12 months after implantation of either bioengineered partial cornea model in rabbit corneas. Clinical results determined by slit lamp and optical coherence tomography were positive after 12 months. Histological and immunohistochemical findings demonstrated that These results suggest that both models are potentially useful to treat diseases requiring anterior cornea replacement, and that HHC may be an efficient alternative to the use of HOC which circumvents the need to generate cornea epithelial cell cultures. Transplantation of a functional tissue-engineered cornea could contribute to the clinical treatment of patients with severe corneal defects . SeveralHowever, the clinical translation potential of most of these strategies is still uncertain and highly dependent on the availability of adequate limbal stem cell sources for the generation of bioengineered cornea models with corneal cells. In this regard, human limbal stem cell cultures raised from limbal biopsies are difficult to obtain, and the procedure is subjected to the risk of causing limbal stem-cell deficiency in the healthy eye .In addition, patients suffering from bilateral limbal stem cell deficiency (LSD) may not be candidates for limbal biopsy and need alternative sources of corneal epithelial cells . For thein vitro  may have the potential to differentiate into cornea epithelial cells for the generation of partial human corneas in vitro . Among tin vitro . In factl source . Althougin vitro and in vivo to determine the differentiation potential of HWJSC and the clinical translational potential of bioengineered corneal substitutes with this source of stem cells.In the present study, we generated partial human orthotypical cornea (HOC) models with corneal cells, and human heterotypical cornea (HHC) models with HWJSC. We then characterized the main stromal and epithelial markers of these bioengineered cornea substitutes both Clostridium histolyticum type-I collagenase  for 6 h at 37°C. Isolated keratocytes were obtained by centrifugation and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 4 mM L-glutamine and 1% antibiotic–antimycotic solution (Thermo Fisher Scientific). Then the limbal tissue was carefully dissected and small explants were cultured directly in Petri dishes with epithelial cell culture medium (2 culture flasks using AmniomaxTM culture medium (Thermo Fisher Scientific). All cell cultures were kept in a cell incubator at 37°C with 5% CO2 using standard cell culture conditions.To generate primary cultures of human cornea stromal and epithelial cells, we used limbal sclero-cornea rings that were discarded after cornea transplant surgery. First, cornea remnants attached to the limbus were isolated by surgical dissection and digested in e medium . HWJSC we medium . First, All methods and experimental protocols were performed in accordance with relevant guidelines and regulations according to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. For the use of human tissues, the project was approved by the local Human Research and Ethics Committee of the province of Granada -PEIBA- (numbers 9/2017 and 3/2016), and all tissue donors or their parents and/or legal guardians provided their informed consent.2 (Sigma-Aldrich). This mixture was rapidly aliquoted in Transwell cell culture inserts with 0.4 μm porous membranes  and allowed to jellify at 37°C for at least 6 h. Then, HOC models were generated by subculturing the previously cultured cornea epithelial cells on top of the stroma substitutes, whilst HHC models were generated with HWJSC instead of epithelial cells. In both models, stratification and differentiation of the epithelial-like cell layer were promoted with an air–liquid culture technique. This technique was performed by lowering the amount of culture medium in the porous inserts, so that only the stromal substitute remained submerged, whereas the developing epithelium was in direct contact with air and received nutrition from the stromal layer  and heterotypical models containing non-corneal cells as the epithelial cell source (HHC) were generated with fibrin-agarose biomaterials with a final agarose concentration of 0.1% as previously reported by our research group . To do sal layer .Finally, bioengineered corneas were subjected to plastic compression nanostructuration . This tein vivo, samples were implanted on the anterior cornea surface of New Zealand laboratory rabbits using the DALK technique. A total of 20 rabbits were included in the study, which were randomly assigned to one of the following groups: (1) four animals were grafted with HOC and euthanatized after 3 months of follow-up, (2) four animals with HOC analyzed after 12 months, (3) four animals with HHC analyzed after 3 months, (4) four animals with HHC analyzed after 12 months, and (5) four non-operated rabbits used as controls.Animal study design: in order to characterize both cornea models Animals were deeply anesthetized by an intramuscular injection of xylazine (5 mg/kg) followed 10 min later by an intramuscular injection of ketamine (25 mg/kg), and local anesthetics drops were administered in the eye. Then the right eye was exposed, and the anterior layers of the cornea  were surgically removed using a surgical microscope. To do that, a manual keratotome was first used to make a circular incision approximately 6 mm in diameter and 200 μm deep in the corneal thickness. Then an air bubble was injected into the central layer of the cornea stroma with a Hessburg-Barron vacuum trephine, and an ophthalmic crescent knife was used to horizontally dissect the most external stromal layers along with the superjacent epithelium. The limbus was preserved in all eyes. After careful debridement of the cornea surface, the bioengineered HOC or HHC was placed on this surface and sutured to the remaining cornea with 10/0 nylon suture stitches. Local antibiotics and anesthetics drops were administered after surgery. To protect the artificial tissue from mechanical damage, the eyelid of the treated eye was closed for 1 week. In all animals, the left eye was not treated and served as a control.Ophthalmic evaluation was carried out every week, and slit-lamp bio-microscopy and anterior segment optical coherence tomography (OCT) was performed at the moment of the euthanasia of each animal . Both eyes were examined in each animal, and major side effects were registered, including tumorigenesis, hemorrhage, rejection or infection. The presence of inflammation was identified by the presence of hyperemia or neovascularization. Then, all eyes were surgically extracted and analyzed histologically as described below.Animal experimentation was approved by the regional ethical committee , ref. number 25/06/2018/099.in vitro HOC and HHC samples corresponding to 1, 2, and 3 weeks of development and in vivo HOC and HHC samples grafted in laboratory animals for 3 and 12 months. Samples were fixed in 3.7–4.0% wt/vol buffered formaldehyde for 24 h at 4°C and dehydrated in ethanol series , cleared in xylene (two incubations of 1 h) and embedded in paraffin (two incubations of 1 h in paraffin melted at 60°C) using an automated Citadel Tissue Processor (Thermo Fisher Scientific). Tissue sections 5 μm thick were obtained and placed on glass slides, dried at room temperature, dewaxed with three incubations of 5 min in xylene and rehydrated with an ethanol series .Histological methods were used to evaluate For histological analysis, tissue sections were incubated for 3 min in hematoxylin , rinsed in tap water for 5 min, stained with eosin (PanReac AppliChem) for 1 min and dehydrated using an ethanol series (3 incubations of 5 min in 96% and 3 incubations of 5 min in 100% ethanol), xylene (3 incubations of 15 min) and cover-slipped using Permount . Tissue sections stained with H&E were used to quantify the number of cell layers in the corneal epithelium. In each sample, the epithelial layer was examined using 600× magnification, and the number of cell layers was determined by three expert histologists.Immunofluorescence assays in tissue sections were performed using previous standardized protocols . Samples2O2 to quench endogenous peroxidase, and antigen retrieval and prehybridization were carried out as described for immunofluorescence. Then, samples were incubated overnight at 4°C in anti-type I collagen (Acris R1038) or anti-vimentin (Sigma V6630) primary antibodies and washed in PBS. Then, peroxidase-labeled secondary antibodies (Vector Laboratories) were used and the signal was detected using a diaminobenzidine (DAB) development kit (Vector Laboratories). In both cases (immunofluorescence and immunohistochemistry), isotype-specific immunoglobulins were used instead of the primary antibodies as negative controls.For immunohistochemistry, tissue sections were treated with 3% HFor scanning electron microscopy (SEM), samples were fixed in cacodylate-buffered 2.5% glutaraldehyde, dehydrated in alcohol series  and dried by the critical point method using a Critical Point Dryer . This method consists in replacing all the ethanol embedding the tissue by liquid carbon dioxide at high pressure, which is then evaporated at low pressure, resulting in a critically dried sample. Then, tissues were sputter-coated with gold-palladium using a Mini Sputter Coater System  and examined a Quanta 200 scanning electron microscope .For transmission electron microscopy (TEM), samples were fixed in cacodylate-buffered 2.5% glutaraldehyde for 8 h, washed three times in cacodylate duffer and postfixed in 1% osmium tetroxide for 90 min. After fixation, samples were dehydrated in increasing concentrations of acetone , embedded in Spurr’s resin and cut into ultrathin sections with an ultramicrotome. For analysis, sections were stained with aqueous uranyl acetate and lead citrate and examined with an EM902 transmission electron microscope .In order to characterize the different cornea models from an optical standpoint, we analyzed the transmittance of each sample and the reduced scattering and absorption coefficients. The inverse adding-doubling method was used to determine scattering and absorption using total reflection and total transmission measurements made with a single integrating sphere, as previously described , 2017. Tpost hoc comparisons between two specific types of corneas  was performed using the Mann–Whitney test. Non-parametric tests were used because the parameters analyzed here did not fulfill the criteria for parametric testing. A Bonferroni-adjusted P-value of 0.001 was considered as statistically significant for the double-tailed tests, since numerous statistical comparisons were carried out at the same time. P-values between 0.05 and 0.001 were considered as marginally significant, meaning that these values would have been considered as significant if the Bonferroni correction had not been applied.Quantitative results obtained for the optical parameters  were analyzed statistically to identify differences among samples. First, a global comparison of all study samples  was carried out using the Kruskal–Wallis test. Then, In vivo analyses of generated HOC and HHC models allowed us to evaluate the safety and biointegration of bioartificial corneas. In this regard, we found no signs of tumorigenesis, hemorrhage, rejection, infection, or other major side effects in any of the animals. Macroscopic examination of the rabbit eyes revealed that HOC and HHC resulted in corneas with some inflammatory signs around the suture stitches, especially 3 months after in vivo implantation. However, inflammation tended to resolve after 12 months. In fact, ophthalmic evaluation of HOC revealed some inflammation around the stitches in 2 out of the 4 rabbits analyzed at 3 months, and 1 out of 4 rabbits at 12 months. For HHC, 2 of the 4 animals showed inflammation at 3 months, and none at 12 months . However, the type of sample was directly related with this parameter, and HOC in vitro samples showed significantly higher transmittance than HHC in vitro samples (P = 1.57 × 10–272). The absorption and reduced scattering coefficients values of HOC in vitro samples were significantly higher than controls (P = 2.31 × 10–5 for absorption and P = 1.81 × 10–39 for scattering), and the same behavior was found for HHC in vitro samples (P = 1.78 × 10–122 for absorption and P = 2.37 × 10–199 for scattering). However, HOC showed significantly lower levels of absorption and reduced scattering than HHC in vitro samples (P = 1.01 × 10–204 for absorption and P = 3.78 × 10–285 for scattering)  . Interesttering) .in vivo, we found a substantial increase in transmittance values. In HOC and HHC in vivo models 3 months after implantation, global mean transmittance was approximately 55 and 63%, respectively, corresponding to approximately 66 and 76%, respectively of the transmittance in control corneas. Therefore, we found an increase of transmittance 12 months after implantation, with transmittance values of approximately 75% for both HOC and HHC, corresponding to 90% of control corneas. Differences between HOC and HHC were marginally significant at this time (P = 0.00719), but both cornea models were significantly lower than controls (P = 1.82 × 10–180 for HOC and P = 2.96 × 10–196 for HHC). Regarding the absorption coefficient, we found that both HOC and HHC had significantly higher absorption coefficients than controls after 3 months of follow-up (P < 1.00 × 10–100 for HOC and HHC), with HOC being higher than HHC (P = 0.00001). However, in vivo samples 12 months after implantation showed absorption coefficients that were lower than those in control corneas, with differences being statistically significant (P < 1.00 × 10–100). Finally, we found that the reduced scattering coefficients of HOC and HHC after 3 months were lower than in vitro samples, although did not reach the low levels of control corneas (P < 1.00 × 10–100 for HOC and P = 4.84 × 10–302 for HHC). After 12 months, reduced scattering values were very similar to control corneas, although statistical differences with controls were detected (P = 1.58 × 10–111 for HOC and P = 2.36 × 10–88 for HHC). At this time, differences between HOC and HHC were marginally significant (P = 0.19009) for the reduced scattering.When HOC and HHC were grafted in vitro construction of human bioartificial cornea substitutes without the drawbacks associated with the use of autologous limbal stem cells , we might hypothesize that the use of MSC in HHC could exert a positive influence in terms of inflammation. Even though future works should be carried out with a larger sample size to confirm or not this hypothesis, the immunomodulatory effects of HWJSC have been previously demonstrated in different settings , and that native HWJSC express high amounts of MSC undifferentiation markers, which decrease upon epithelial differentiation coinciding with a positive expression of specific differentiation markers  showed positive expression in corneas grafted in rabbits, and very low expression in rabbit controls. Although this supports the possibility that the cells found in corneas grafted in vivo were human, survival of the grafted epithelial cells should be confirmed in future studies using FISH analysis or other highly sensitive techniques  and the highest levels of light absorption and reduced scattering, especially in the first weeks of development, and suggest that the corneal epithelium differentiation level of these samples was low. Remarkably, the expression levels of collagen type I also tended to be low in corneas maintained in vitro, suggesting that an appropriate organization of the stromal fibers is crucial for corneal transparency . The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics and Animal Experimentation Committee of the University of Granada and the Andalusian Directorate of Agricultural Production (CEEA).IG, JC-A, MM-P, and MA carried out the artificial tissue development, data analysis, and manuscript preparation. CG-G and JC-A carried out the animal studies and obtained the clinical results. AI, JC, and MP were responsible for the optical analyses. MM was involved in immunofluorescence analyses in all samples. CC was responsible for TEM microphotographs. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


At the early stage of implantation, the immunological indexes such as Immunoglobulin G, Immunoglobulin M concentration and CD4 cells’ population of allogenic bone significantly increased in compared with those of autologous bone and artificial bone. Although the repair process of artificial bone was relatively inefficient than autologous bone graft, the low immunotoxicological indexes and acceptable therapeutic effects endowed it as an excellent alternative material to solve the problems with insufficient source and secondary trauma of autogenous bone.Repair and reconstruction of large bone defect were often difficult, and bone substitute materials, including autogenous bone, allogenic bone and artificial bone, were common treatment strategies. The key to elucidate the clinical effect of these bone repair materials was to study their osteogenic capacity and immunotoxicological compatibility. In this paper, the mechanical properties, micro-CT imaging analysis, digital image analysis and histological slice analysis of the three bone grafts were investigated and compared after different time points of implantation in rat femur defect model. Autogenous bone and biphasic calcium phosphate particular artificial bone containing 61.4% HA and 38.6% β-tricalcium phosphate with 61.64% porosity and 0.8617 ± 0.0068 g/cm Bone defect could easily lead to non-union and loss of bone functions, which greatly affects the life quality of patients. Orthopedics is usually used to stimulate fracture healing and reconstruct the lost native anatomy , 2. For Allogenic bone substitutes had relatively extensive sources and were easy to process and store. There was no risk of donor site injury as autogenous bone transplantation. Allogenic bone had an open, porous and reticulated physical structure similar with that of autogenous bone, which was conducive to the vascularization of bone after implantation. It is reported that the healing time of fracture implanted with allogenic bone substitute was close to that of autogenous bone . Based oin vivo, it could interact with body liquid and support the formation of new bone tissue. On the other hand, artificial bone grafts might adsorb and desorb functional proteins, resulting in changes in protein conformation [in vivo and cause immunotoxicity [Artificial bone grafts such as hydroxyapatite (HAp), β-tricalciumphosphate (β-TCP) and bioactive glasses represented promising alternatives because they did not have some of the drawbacks mentioned above. After the implantation of artificial bone grafts ormation , 20–25. toxicity . Besidestoxicity , 28. Somtoxicity , 30.Calcium phosphate bioceramics are important type of artificial bone materials. Porous biphasic calcium phosphate bioceramic had osteoinducing property, and could promote the regeneration of defected bone, enhance the bone integration and promote the new bone formation . It had 3 density (d ≤ 2mm) were purchased from National Engineering Research Center for Biomaterials . Enzyme-linked immunosorbent assay (ELISA) kit for Immunoglobulin G (IgG) and ELISA kit for Immunoglobulin M (IgM) were purchased from Cloud-Clone Corp (USA). Fluorescein isothiocyanate (FITC) mouse Anti-Rat CD4 and PE mouse Anti-Rat CD8a were purchased from BioLegend (USA). Roswell Park Memorial Institute 1640 (RPMI 1640) and fetal bovine serum (FCS) were purchased from HYCLONE (USA).Biphasic calcium phosphate ceramics particles with 61.64% porosity and 0.8617 ± 0.0068 g/cmExperimental rats were purchased from Chengdu Dashuo Experimental Animal Co., Ltd (China). All animal studies were approved by the Sichuan University Medical Ethics Committee. All animal procedures were performed in accordance with the guidelines for care and use of laboratory animals of Sichuan University.Autogenous bone was collected from anesthetized animals. A longitudinal on the skin over the iliac bone area was created, then the soft tissue attached to the iliac bone was separated. The iliac bone tissue was separated and removed using bone scissors and hemostatic forceps, and was used for transplantation directly. Allogenic bone substitute was prepared according to a previously published literature by destroying or even removing rat antigens through freezing in a refrigerator at −80°C for 2 weeks . The artThe surface morphology and the crystal structure of materials were analyzed by scanning electron microscope  and X-ray diffraction (XRD) . The SEM samples were prepared. For XRD, scanning range was from 20° to 80° with step size of 0.04° and step time of 1 s. For microscopic observation of autogenous bone and allogenic bone substitutes, samples were prepared by gradient dehydration in series concentration of ethanol and critical-point drying.in vivo study, murine femur defect model was chosen to estimate bone formation. The experimental animals were divided into three groups: autogenous bone group, artificial bone group and allogenic bone group, with 30 SD rats in each group . The time point of test was 2, 4, 12, 26 and 40 weeks, respectively. There were six parallel animals in each time point in each experimental group. After the mice were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/ml), the femoral region was sterilized with iodine. The subcutaneous tissue was bluntly separated and the femur was exposed along the two muscle bundles. The periosteum was cut with a surgical knife. A circular hole of 5 mm diameter was drilled near the knee joint at the distal end of the femur to reach the bone marrow cavity. The experimental material was implanted into the hole according to the ratio of 1.2 g/kg body weight [For y weight .The animals were anesthetized at 4, 12 and 40 weeks for X-ray examination. The test parameters are 52 kV, 7.5 mAs, with grids, automatic exposure closure. X-ray film of both hind limbs was taken. Bone repair at bone defect site was observed, such as bone junction, bone healing and bone modeling.After euthanasia of the experimental animals at 4, 12 and 40 weeks, the femurs were removed and the soft tissue on the femur was removed. The femur was numbered and inserted into the cylindrical foam. The femoral implant was then scanned by conical beam micro-focal X-ray bulb tube micro-CT scanner. The scanning parameters were the resolution size of the scanning was 22 μm; the rotation angle was 360°; the rotation angle increment was 0.4°; 55 kV, 72 μA, 4 weeks.Five SD rats were euthanized at 4, 12 and 40 weeks after implantation. The femur was removed and the implanted area was cut off. The femur was washed with PBS three times. All specimens were fixed in 3% formaldehyde solution for 2 weeks, decalcified in 10% EDTA solution and the media was changed every 3 days. After decalcification, dehydration, paraffin embedding, slicing, dyeing and other tissue treatment processes were used to prepare tissue sections. Tissue sections were 5 μm thick and stained with hematoxylin−eosin.6 cells/ml. The above lymphocyte suspensions were stained with FITC mouse anti-rat CD4 (Lot: 5299960) and PE mouse anti-rat CD8a (Lot: 6175730) labeled with BioLegend fluorescence, and then analyzed by flow cytometry.The test of IgG, IgM was performed as following. The blood of experimental animals was taken by tail vein cut off to test the immune indexes. Peripheral blood of rats was collected in a 1.5 ml centrifugal tube. In order to prepare serum, peripheral blood of rats was centrifuged for 10 min at 1000 g. The upper serum was collected and stored at −20°C. The serum was placed at room temperature for 30 min before use, then tested by ELISA method according to the operation instructions of ELISA kit. At the end of the immune period (4 and 26 weeks), the rats were euthanized, and the spleens were taken aseptically. Each sample was grinded to prepare a single cell suspension. Cell suspension was collected. RPMI1640 containing 10% FCS was added to adjust the cell concentration to 2 × 10At 4 weeks, the femurs were dissected and cut into cylinder of 5 mm diameter and 10 mm height by a hard tissue slicer. The precise dimensions of samples were measured by digital calipers. The sample was placed between the two indenters of the test machine and was compressed at a rate of 5 mm/min and compressive strength of each specimen was calculated , 39. Thet-test. The significance level of statistical test was set to P < 0.05, **P < 0.01 and ***P < 0.001.All data in the experiment were expressed by mean (+SD). The experimental data were compared by AVONA method and The macrograph of autogenous bone, allogenic bone and artificial bone were shown in The reconstructed 3D models by micro-CT of the three samples were displayed in To evaluate bone regeneration inside the grafts, a cylindrical region of interest in each group was chosen in each sample for quantitative analysis. The detailed information on structural parameters was shown in To further detect the bone regeneration throughout the three bone grafts, cross-sections of the grafts were assessed by histological observation. As shown in At 12 weeks, reticulated bone trabeculae in the implantation orifice and medullary cavity was observed in autogenous bone group, some of which had been calcified into bone tissue, and a large number of bone marrow cells around the bone tissue were seen under high power microscopy . SimilarThe compression test results of different implant samples at different implantation time points were shown in The change of antibody concentration was the main manifestation of immune response. IgG, IgM and other antibodies were immunoglobulins that could specifically be combined with antigens and played an important role in the immune response of biomaterials . As showThe percentage of CD4 and CD8 lymphocyte expression was one of the important indicators of immune system function, especially the immune regulation function of organism. Flow cytometry results of the three groups indicated that the CD4 and CD8 expression of spleen lymphocytes in three groups increased with time extension . The quain vivo for a period of time, all these materials promoted the formation of new bone. Among them, autogenous bone and artificial bone substitutes had similar strong ability to repair bone defects. However, the autogenous bone was taken from the iliac bone of experimental animals. It had been reported that the increased incision, prolonged operation and anesthesia time damaged bone structure and functional stability of donor site, and might result in donor site complications [The main purpose of clinical bone defect repair is to reconstruct the bone structure and restore the physiological function of bone. In this study, a rat femur defect model was established. Autogenous, allograft and artificial bone substitutes were implanted in the defect sites; their bone regeneration and immunotoxicity were investigated. The X-ray, gross observation, micro-CT, histological observation and mechanical property results showed that, after implanting ications , 43. In ications . Hence, in vivo, leading to cell loss or cell death [in vivo [On the other hand, allogeneic bone grafts exhibited worse repairing ability compared with other two materials. Besides, higher level concentration of immunoglobulins  was found in allogeneic bone group, which was consistent with the results of histological observation. The relatively worse repairing ability of allogenic bone was probably due to the immunotoxicity of the material. Because of the relationship between immune rejection and immunotoxicology, the main problems in clinical practice of allogenic bone were delayed healing of bone, unsatisfactory vascularization, low healing strength and non-union . The immll death . The tra[in vivo , 20, 25.in vivo, especially at the early stage of implantation, the immunological indexes such as IgG, IgM concentration and CD4 T cells population of allogenic bone significantly increased to the degrees that were significantly higher than that of autogenous/artificial bone. Even after deep freezing, the antigenicity of allogenic bone still remained difficult to totally eliminate [in vivo and did not produce humoral immune toxicity reaction [The analysis of CD4 and CD8 positive expression of lymphocyte in spleen and other immune organs by flow cytometry was used to explore whether medical devices contacted with organism had potential ability to affect the immune system function of organism , 56. At liminate . These rreaction , 59.in vivo. The results showed that artificial bone substitute, which contained 61.4% HA and 38.6% β-tricalcium phosphate with 61.64% porosity and 0.8617 ± 0.0068 g/cm3 density (d ≤ 2mm), had strong bone repair ability, similar with that of autogenous bone graft. On the other hand, the bone repair ability of allogenic bone graft was relatively poor, even after antigen removal by freeze−thawing treatment, probably due to immunotoxicological reaction of materials. This study provided experimental basis for the selection of bone repair materials for clinical use. Although the repair process of artificial bone was relatively inefficient than autologous bone graft, the low immunotoxicological indexes and acceptable therapeutic effects endowed it an excellent alternative material to solve the problems of insufficient source and secondary trauma of autogenous bone.In this study, the osteogenesis effect of autogenous, allogenic and biphasic calcium phosphate artificial bone substitutes on rat femur defect model were investigated REGBIO online.This work was supported by National Key Research and Development Program of China (2018YFC1106800) and Sichuan province key research and development project (20ZDYF0191). The authors greatly appreciated Cheng Li from Analytical & Testing Center of Sichuan University for her assistance in Micro-CT measurement. Conflict of interest statement. The authors declare no competing non-financial/ﬁnancial interest. rbaa040_Supplementary_DataClick here for additional data file.