Archivum Immunologiae et Therapiae Experimentalis (AITE) was founded in 1953 by Ludwik Hirszfeld, a world famous Polish physician and scientist in the field of microbiology and immunology. Initially, AITE was published in Polish, but within a few years, it changed to English to increase the range and number of international readers. In its over 65 year history, AITE has had several Editors and a number of Publishers. In the period 1977–1991, AITE was listed in the system of scientific information Current Contents/Life Sciences, but for several years, its impact on the international readership of the Journal was negligible. The political and economic crisis in Poland in late 1980s led to serious delays in printing of successive AITE issues, so the Journal was removed from the Current Contents. Year 1991 was a turning point for the Journal, guided since then by prof. Dubowska-Inglot, who changed its image and format, and allowed acceptance of review articles. In 1999, prof. Górski became the Editor-in-Chief, giving a new impulse for further development of the Journal. In a consequence, AITE was accepted to Science Citation Index Expanded (in 2001) and to Institute for Scientific Information Master Journal List (in 2002). Eventually, AITE has evolved to become a truly international, multidisciplinary journal, publishing original articles, and reviews relating to basic and clinical immunology, experimental therapy, immunogenetics, transplantology, microbiology, immunochemistry, as well as bioethics. Currently, AITE is cited in a number of major scientific information databases. Since 2011, the Journal is published by Springer Publishing House, it has achieved international recognition with its latest impact factor (for 2017) of 3.018. AITE, whose Editors are professors of Hirszfeld Institute of Immunology and Experimental Therapy, strengthens the status and position of the Institute as one of the leading scientific institutions in Poland. Archivum Immunologiae et Therapiae Experimentalis (AITE) was founded in 1953 in Wrocław (Poland) by Ludwik Hirszfeld, a world famous physician, serologist and immunologist. At the time in the postwar Poland, a great demand existed for establishment of a new journal due to a remarkable progress in the field of immunology and related medical sciences. The Journal was initially edited in Polish, but after some years, English was introduced for all articles to increase the range and number of potential international readers. AITE in its 65-year history had several Editors-in-Chief : Henryk Makower (1953–1955), Stefan Ślopek (1956–1987), Jerzy Giełdanowski (1988–1991), Anna Dubowska-Inglot (1991–1998) and since 1999 until now—Andrzej Górski. The Journal was initially published by Polish Scientific Publishers , then by Polish Medical Publishers , Ossolineum , Medical Science International , Birkhäuser  and finally Springer . Figure At the beginning AITE was published solely in Polish, and from 1961, the articles are in English. Although AITE was added to Current Contents/Life Sciences (CC/LS) system in 1977, for several years its impact on the international readership of the Journal was low. The political and economic circumstances in Poland in the late 1980s led to nearly a year delay in printing of AITE issues, which finally resulted in deselecting from CC/LS in November 1991. The Journal’s condition was poor—the scientific value of articles, as well as the reviewing procedures, was not adequate; in addition, the bureaucracy and poor management of the then publishing house negatively affected editorial work. In consequence, the impact factor (IF) for 1988 was the lowest in AITE history (0.104). Table Autoimmunity and Inflammation  changing its size to A4, (2) introducing color figures and review articles  and (3) introducing a two-column format. Fortunately, the respective issues of the Journal started again to be printed duely on time. In 1999, after passing away of prof. Dubowska-Inglot, prof. Andrzej Górski was appointed a new Editor-in-Chief, giving a further impulse to improve the quality of the Journal. A new approach to achieve a higher scientific status and international position of AITE involved the procedure of inviting review articles from renowned potential authors. In 2000, the IF of AITE was still modest (0.413) although four times higher than in 1988. AITE started to be much better recognized on the international publishing market. Taking the chance of a higher influx of review articles, AITE was able to publish, in collaboration with Kluwer Academic Publishers, two monothematic books: After collaboration with Ossolineum AITE was taken up in 2004–2005 by Medical Science International (Warsaw), which used more up-to-date editorial technologies, this change resulted in higher IF 1.00 in the year 2005. Such progress attracted the interest of Birkhäuser Publishing House (at that time filia of Springer), which we signed a publishing agreement with starting 2006. In consequence, in the years 2006–2010 AITE was published by Birkhäuser. Thanks to association with this professional, international publishing house, the Journal accomplished a tremendous progress by achieving IF 2.385 in 2010. In 2011 we started the collaboration with Springer which brought further increase of AITE citation (IF 3.176 in 2014). This constant increase of AITE scientific level let the Journal rank three times first: in 2011 (IF 2.541), 2013 (IF 2.818) and 2014 (IF 3.176) among Polish scientific journals, present in ISI Master Journal list. Following a brief decline in IF value in the years 2015–2016, AITE received IF 3.018 (2017), which is close to the highest value in the history of the Journal (IF 3.176 in 2014). We are proud to be successful in continuously attracting authors from prestigious universities, like: Harvard, Oxford, Yale, Tokyo, Athens, Houston, Hong Kong, Edinburgh, Osaka, McGill, Karolinska Institutet and many others. AITE articles are being cited in the leading scientific journals, such as: Science, Nature, Journal of Immunology, Journal of Experimental Medicine, PNAS, Oncotarget, Frontiers in Immunology, Frontiers in Microbiology, Scientific Reports, PLoS One, etc. In 2011 the Editorial Manager (EM) system was introduced to our editorial practice which is a rule in the most modern scientific journals. The system substantially facilitates editorial procedures and manuscript processing. AITE is currently cited in the following basic science information systems: Science Citation Index Expanded (SciSearch), Journal Citation Reports/Science Edition, PubMed/Medline, SCOPUS, EMBASE, Chemical Abstracts Service (CAS), Google Scholar, CSA, CAB International, Academic OneFile, AGRICOLA, Biological Abstracts, BIOSIS, CAB Abstracts, Gale, Global Health, INIS Atomindex, International Bibliography of Book Reviews (IBR), International Bibliography of Periodical Literature (IBZ), OCLC, Journal Citation Reports/Science Edition, SCImago and Summon by Serial Solutions.The policy of AITE assumes recruitment of the best quality manuscripts irrespectively of affiliation, country of origin and scientific output of a potential author, however, manuscripts from renowned authors and institutions have our preference. First, the manuscripts are subjected to the editorial in-house evaluation and these outside the scope or of lower priority are not processed further. The Editors, as a rule, do not accept case reports. As a next step, the manuscripts are subjected to peer review by at least two foreign experts in the field, although some articles require evaluation by more referees. Invited articles are published free of charge. The Journal accepts both original and review articles, although from Editorial Office point of view, the latter are preferred due to a higher readership and citation. In practice, the number of reviews and original articles per issue are roughly comparable (1:1). The policy of AITE also stipulates that new members of the Advisory Board are recruited from new authors and their exceptional contribution to the scientific level of the Journal is a requirement. The list of Advisory Board is a subject to modification every 3 years.The Journal of Immunology. Recently a remarkable increase of the number of new journals is evident, mostly published online, which leads to a dramatic competition on the market. Luckily, these newly generated journals are not attractive enough due to lack of established IF and absence, at the beginning, in the scientific information systems. In this respect, the tradition of AITE (founded in 1953) and its excellent history of attaining the international status, represent an advantage for the Journal. About 50% of the articles, published in AITE, are currently (2018) accessible to readers free of charge.The gaining of the good quality articles is a serious challenge. Apart from spontaneously submitted manuscripts, of which many may not meet the Journal’s requirements, we try to focus on a category of “invited reviews”. Such invitations are usually aimed at authors published in high-quality journals, for example, The Journal of Immunology suffered a drop in its IF from 7.065 (in 2001) to 4.539 (in 2017). Although new attractive titles in the field of immunology emerged, due to a constant progress AITE found a firm place among important immunological journals (IF in parenthesis), such as: Immunobiology (2.720), Cellular Immunology (3.172), International Immunopharmacology (2.956), PLoS One (2.806), Acta Pharm Sinica (3.223), Basic and Clinical Pharmacology and Toxicology (3.176) or Scandinavian Journal of Immunology (2.256). These comparisons clearly show a leading position of AITE among immunological journals in Central and Eastern Europe  varied depending on IF gained in a given year. AITE ranked in the years 2010–2012 in quartile Q3, in quartile Q2 in the years 2013–2014, in quartile Q3 in the year 2015 and in quartile Q4 in the year 2016. The IF 3.018 for the year 2017 moved AITE to quartile Q3 again. It is also obvious for us that because of high competition on the market recently we may observe serious shifts in a position of previously leading journals, e.g., The most difficult part of our editorial work is a proper selection of articles which would be interesting to the readers. This is reasonably easy for some types of review articles but sometimes we can not foresee a potential “success” of a given article. A case in point: it happened that we published several articles from one research group and they were very poorly cited causing our objection to accept another manuscript from the same laboratory. In addition, the opinions of the reviewers regarding the newest paper were unpromising. However, to our surprise, the article had quite high citations. Another important issue regarding acceptance of articles for publication is that it turned out to be risky to publish articles from conferences presenting subjects not directly associated with the publishing scope of AITE. In these cases the articles were not at all or very poorly cited which, in consequence, negatively affected the Journal’s IF.It is of course clear that the significance of IF value for a scientific journal should not be overestimated. A widely spread opinion prevails that IF does not actually reflect the real scientific value of a journal Callaway  and whatNevertheless, in the Polish Ministry of Science and Higher Education Pilc  system oAITE Editors pay particular attention to maintaining high ethical standards of the Journal and thus the Editors decided not to publish their own articles in AITE. Solicitation, expressed by Editorial Office, to cite AITE articles by the authors of accepted papers, , is totally unthinkable. In consequence, the percentage of self-citations in AITE articles is amazingly low , published by Clarivate Analytics since 1975, monitor the journals towards unexplainable high citations and, in the cases of dishonest procedures of acquiring citations, remove those journals from JCR databases for 1 year. AITE, in its 65 years history, has always been blameless regarding this issue.The Editorial Office is also aware of potential plagiarism, and therefore, the submitted manuscripts are monitored using an anti-plagiarism computer program. Ethical responsibilities of the authors are included in the Instructions to authors and require a statement of: (1) novelty of the presented results not published before; (2) potential conflicts of interest; (3) agreement of local Ethics Committee regarding the research involving human participants and/or experimental animals and (4) informed consent of patients and volunteers donating biological material. The In the last 26 years the scientific value of AITE has acquired good international level. Our Advisory Board currently consists of 39 members, 26 of whom (67%) come from the foreign scientific institutions. To complete the current, overall image of AITE below we present some characteristic data:All manuscripts, submitted to AITE and accepted by Editors, are subject to peer review procedure. As a rule we look for reviewers abroad and pay due attention to their thorough opinions and subsequently to Authors’ responses, who are mostly satisfied with these comments. This is reflected in their positive attitude, e.g., “The reviewers’ comments have been most helpful to improve the quality of our manuscript”, “Thank you very much for this opportunity to publish in AITE”, “We express our gratitude to Reviewers for their comments that helped us to improve the quality of the article”. We fully understand that thanks to the Reviewers’ work the scientific quality of the Journal constantly improves.Table AITE publishes yearly ca 45–50 articles; from the Table It should be noted that some 25 years ago the influx of the manuscripts being very poor, the Journal did not reject a single article. Nowadays the number of manuscripts submitted is considerably higher and we have a real choice as to which manuscripts should be published.As mentioned above, citation is the best measure of the scientific value of an article and the journal, where it was published. The best-cited AITE articles are as follows :179 citations—Zhang et al. ,147 citations—Shi and Wei ,147 citations—Varnum and Ikezu ,142 citations—Bujak and Frangogiannis .136 citations—Kobayashi et al. .AITE, in its 65 years of publishing history, had so far 13 articles cited more than a hundred times and 43 articles were cited at least 50 times. When an article is cited in a scientific publication, it is a clear indication that it was interesting and significant for the scientific world. In consequence, individual citations influence IF of a journal.Another measure of interest in a journal’s articles, although more relative, are full-text down loadings from the website of a journal. Based on AITE Publisher’s Report  these fuin 2010 40,028 requestsin 2011 44,560 requestsin 2012 45,443 requestsin 2013 55,413 requestsin 2014 49,484 requestsin 2015 52,674 requestsin 2016 86,744 requestsin 2017 109,159 requestsIn our opinion the number of the full-text article requests is high and means that AITE articles are interesting to the readers. Moreover, the number of these down loadings is constantly increasing, which is very promising for the Journal.Figure http://www.springer.com/5. The navigation bar includes Online first articles, where there is a set of papers accepted for publication but not yet printed, with a given DOI number which already enables citation.Currently AITE is published in two forms: printed version (ISSN 0004-069X) and electronic version (ISSN 1661–4917); the latter can be reached under the address Editorial OfficeEditor-in-Chief Andrzej Górski since 1999deputy editor Hubert Krotkiewski since 1991deputy editor Michał Zimecki since 1991editorial assistant Anna Steć since 1990Our plans for the nearest future assume continued and fruitful collaboration with Springer Publishing House. Our efforts will also be aimed at achieving a complete coverage of the Journal’s budget  by adjusting the page-charges to the advancing scientific status of the Journal, which we are determined to maintain on the highest possible level.In December 2016 the Editorial Team of the Journal received a scientific award of the President of Polish Academy of Sciences, professor Jerzy Duszyński, for “earning a notable international prestige for the Journal due to the high publishing standards over many years of its publication”. We do hope this award will strongly raise the international position of the Journal in the coming years.


Molecular Oncology will stay at the forefront of all developments in the area of oncology by providing a flexible platform for European and International cancer research.May 2020 be a rewarding year of change. The recent commitment of the European Commission and policymakers to fight cancer in partnership is expected to bring forward the changes long required in the field.  Nevertheless, we have also experienced a loss, as José Manuel Moreira, who has worked as Editorial Manager of Molecular Oncology for 12 years and was lately acting as Reviews Editor, decided to leave the journal to concentrate on his duties as Full Professor at the University of Copenhagen. We very much appreciate the work José has done for the journal, since we would not be where we are today without his efforts. We wish him all the best in his future activities.Last year, Molecular Oncology achieved several landmarks. Increased recognition of the standing of the journal has driven the submission of around 1200 manuscripts in 2019, and, with the valuable contribution of the journal’s Section Editors, we have published over 150 papers, carefully selected for their novelty and quality. Among these, we are proud to showcase two thematic issues: one on Molecular Oncology, starting on January 1st, 2020. I am delighted to welcome Kevin to the journal team. Kevin is an internationally renowned cancer biologist working in the areas of cell death, metabolism and autophagy, with a strong focus on both basic and translational cancer research, which are critical priorities for the journal. Also, he has extensive publishing experience gathered over the years in senior editorial positions  meetings and Gordon conferences. He has also been involved in the organization of several high‐profile scientific conferences, acting as Chair of the 2019 EMBO Conference on Autophagy, The Beatson International Cancer Conference and ‘Genes and Cancer’ – the UK’s longest‐running cancer conference. As a result of these collective achievements and activities, Professor Ryan has received several awards, most notably the ‘EACR Cancer Research Award’ – a highly revered award of the European Association for Cancer Research – and the 2012 Tenovus Medal, which is presented annually to a scientist with a Scottish link. Professor Ryan is also an elected Fellow of the Royal Society of Edinburgh.Autophagy, Commissioning Editor on the FEBS Journal and Deputy Editor on Oncogene. Professor Ryan is excited to be joining Molecular Oncology, which – being an Open Access journal of FEBS Press – serves the scientific community through both the broad dissemination of cutting‐edge research and the investment of its full income on the funding young researchers.Indicative of Professor Ryan’s standing in the field are his memberships in scientific advisory boards and journal editorial boards. Notably, he has served as Associate Editor on As Joint Editors‐in‐Chief, Kevin and I will work closely with the Section Editors, Senior Editors and the new Editorial Manager to oversee the direction, scope and editorial policy that will guarantee the sustained quality of the journal. Moreover, given the increased number of submissions, one of our main priorities will be to ensure the fast handling of incoming manuscripts. Also, we plan to introduce several new article types in order to provide an extended platform for basic/preclinical, early clinical and prevention research, computational analyses of clinical/prevention and biological data, science policy initiatives and more. To start with, we have just introduced Commentaries to initiate discussions on the implications of selected research articles published in the journal (see Commentary in this issue). Commentaries are short (max. 1000 words) articles that highlight advances in knowledge and specific features of selected papers published in the journal.Molecular Oncology will stay at the forefront of all developments in the area of oncology by providing a flexible platform for European and International cancer research.I am looking forward to this year, with the assurance that it will be a rewarding year of change. Importantly, the recent commitment of the European Commission and policymakers to fight cancer in partnership by sharing expertise, resources and data is expected to bring forward the changes long required in the field.


Journal of Ovarian Research [Pursuant to a decree of the Tribunal of Catania, First Civil Section, LJ Pharma SRL obtained an ex Parte order. The Judge orders BioMed Central Ltd, in the person of the legal representative pro tempore to implement the remedy of the retraction notice on the study, signed by Isabella and Raffone, “Does Ovary need d-chiro-inositol?”, published in 2012 in the specialized journal


CbGBE from Caldicellulosiruptor bescii and PhGBE from Pyrococcus horikoshii, which belong to the glycoside hydrolase family 13 and 57 respectively, were cloned and expressed in Escherichia coli. Two GBEs were identified to have α-1,6 branching activity against various substrates, but substrate specificity was distinct. Starch was modified by two GBEs and their in vitro digestibility and structural properties were investigated. Short-branched A chains with a degree of polymerization (DP) of 6–12 increased with CbGBE-modified starch, increasing the proportion of slow digestible and resistant starch (RS) fractions. PhGBE-modified starch resulted in an increase in the RS fraction only by a slight increase in part of A chains . Compared to the proportion of control not treated with GBE, the proportion of α-1,6 linkages in CbGBE- and PhGBE-modified starch increased by 3.1 and 1.6 times. 13C cross polarization/magic angle sample spinning (CP/MAS) NMR and XRD pattern analysis described that GBE-modified starches reconstructed double helices but not the crystalline structure. Taken together, CbGBE and PhGBE showed distinct branching activities, resulting in different α-1,6 branching ratios and chain length distribution, and double helices amount of starch, ultimately affecting starch digestibility. Therefore, these GBEs can be used to produce customized starches with controlled digestion rates.Two thermophilic 1,4-α-glucan branching enzymes (GBEs),  Starch is the most abundant and important polysaccharide consumed as an energy source by humans. Starch is divided into three categories based on its rate of digestion to glucose units in the small intestine: rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) . SDS is Enzymatic modification is one of the preferred methods of producing modified starch with high SDS and RS contents. To reduce the enzymatic hydrolysis of starch and achieve low-GI values, enzymes like pullulanase, isoamylase, maltogenic amylase, α-glucanotransferase, amylosucrase, and glycogen branching enzyme have been applied either individually or in pairs. These enzymes catalyze the partial hydrolysis and debranching, or elongated amylose/amylopectin (AM/AP) chains, resulting in low-GI starch ,5,6,7,8., EC 2.4.1.18) is a starch-modifying enzyme that catalyzes the formation of a new branching point with an α-1,6 glycosidic linkage after cleaving α-1,4 glycosidic linkages [1) and a decreased number of long branched chains (B2 and B3) and form highly branched α-glucan- and AP-like clusters [The 1,4-α-glucan branching enzyme -transglycosylaselinkages . GBEs belinkages . Althouglinkages , the sublinkages ,13. Theslinkages . Some stclusters ,15,16. Tclusters ,15,16.Pyrococcus horikoshii OT3 (PhGBE) in Escherichia coli and successfully solved a three-dimensional structure, allowing the determination of their branching activities and active sites [To improve the reaction activity between mesophilic enzymes and starch, starch-modifying enzymes are applied to solubilized starch after heating (gelatinization) and subsequent cooling to the optimal enzyme reaction temperature . Howeverve sites .PhGBE and GH13 CbGBE from Caldicellulosiruptor bescii, were compared and modified starches were produced by using these two GBEs. In vitro digestibility of the GBE-modified starches was assessed and the structural properties that included branch points, side chain length distribution, and contents of double helices were explored. The findings clarified how these structural parameters are related to starch digestibility and identified the effects of the GBEs belonging to different family (GH13 vs. GH57).In the present study, the enzymatic properties of two thermophilic enzymes, GH57 Aspergillus niger ; and a glucose assay kit (GAGO20) were purchased from Sigma-Aldrich . Isoamylase (Lot 130103a) from Pseudomonas sp.  was obtained from Megazyme International Ltd. . All other chemicals were of reagent grade and were purchased from Sigma-Aldrich.Maltooligosaccharides (degree of polymerization (DP), 1−7), AM (A-0512), and AP (A-8515) from potato starch; normal corn starch (S-4126), high-AM corn starch (A-7043), pancreatin , and amyloglucosidase from ph_RS06575 (accession number WP_010885475) and athe_RS02765 (accession number WP_015907139) was performed using P. horikoshii OT3 and C. bescii DSM6725 genomic DNA as templates, respectively. The phGBE-specific primers PhGBE-F (5′-TTCATATGAAAGGATACCTAACGTTTGTTCTACA-3′) and PhGBE-R (5′-AGCTCGAGAAGGGTTGATTTCTTCTTTTC-3′) containing NdeI and XhoI restriction enzyme sites (underlined) and the CbGBE-specific primers CbGBE-F (5′-TGGAATTCATGATAAAAAAAGTAAAATCTAC-3′) and CbGBE-R (5′-TCCTCGAGTATCTGAATATTGTTGTC-3′) containing EcoRI and XhoI sites were used. PCR was performed using 2.5 U of PrimeSTAR polymerase  under the following conditions: 25 cycles at 98 °C for 10 s (denaturation), 55 °C for 5 s , and 72 °C for 2 min (extension). The 1680-bp (PhGBE) and 1950-bp (CbGBE) PCR products were purified using a PCR purification kit  and then digested using NdeI and XhoI or EcoRI and XhoI, respectively. The digested DNA fragments were separately ligated into the pET29b(+) vector , which was digested using the same restriction enzymes. The resulting expression vectors pET29b::PhGBE and pET29b::CbGBE encoded PhGBE and CbGBE with a C-terminal hexa-histidine tag, and its sequence was confirmed by DNA sequencing. The pET29b::PhGBE and pET29b::CbGBE were transformed into E. coli BL21(DE3), and the transformants were selected as described previously [To detect GBE genes, PCR amplification of eviously .E. coli expression system and purified using Ni-NTA agarose  [w/v) SDS-PAGE using DokDo MARK .The recombinant proteins were produced in this ont, UK) . Proteinont, UK) . The purw/v) amylose in 50 mM sodium acetate buffer (pH 6.0) was incubated with 0.2 µg of the purified PhGBE and 0.09 µg of the purified CbGBE separately at 60 °C for 15 min. The reaction was terminated by adding 50 µL of 1 M HCl and neutralized by adding 50 µL of 1 M NaOH. Aliquots of 100 µL were mixed with 900 µL of iodine solution (0.02% (w/v) I2 and 0.2% (w/v) KI), and absorbance was measured at 660 nm. One unit (U) of GBE was defined as the amount of enzyme that caused a decrease in absorbance of 0.01 optical density (OD) units per min under the assay conditions [The iodine assay was used for measuring branching activity. The assay is based on monitoring the decrease in absorbance of the glucan–iodine complex resulting from amylose branching. The reaction mixture (50 µL) containing 0.1% (nditions . The optw/v) of each substrate  in 50 mM sodium acetate buffer (pH 6.0) was incubated with 5 U of PhGBE or 2 U of CbGBE at 60 °C for 12 h. After adding 10 U isoamylase in 50 mM sodium acetate buffer (pH 4.0) to the reaction mixture in equal volume, the total reaction mixture (100 µL) was incubated at 40 °C for an additional 12 h, and the reaction was stopped by boiling for 10 min. After centrifugation , the supernatant was spotted on a silica gel 60 F254 plate . The plate was placed in a chamber equilibrated with a solvent mixture containing isopropyl alcohol/ethyl acetate/distilled water  and developed by dipping it into a solution containing 0.3% (w/v) naphthol and 5% (v/v) H2SO4, followed by baking at 120 °C for 10 min to identify the spots.A reaction mixture (50 µL) containing 0.1%  in 50 mM sodium acetate buffer (pH 6.0) was incubated with 5 U of PhGBE or 2 U of CbGBE at 60 °C for 20 h. The products were analyzed using a high-performance anion-exchange chromatography (HPAEC) column with a pulsed amperometric detector . The detailed method was described previously [To determine the action pattern of eviously . The sidw/v) in 50 mM sodium acetate buffer (pH 6.0) containing dimethyl sulfoxide  was boiled with stirring for 30 min. After cooling the mixture to 60 °C, PhGBE or CbGBE (2000 U and 5000 U) was added to the starch slurry and incubated at 60 °C for 20 h. The reaction was stopped by heating in boiling water for 30 min. After removing the insoluble materials by centrifugation, the solution was precipitated by adding three volumes of absolute ethanol. The enzyme-modified starch was precipitated by centrifugation  at 4 °C and washed four times with distilled water. The modified starch was freeze-dried and pulverized. Starch that was incubated under the same conditions without GBE treatment was used as a control.A reaction mixture containing normal corn starch  spectrometer . Each sample (40 mg) was dissolved in 2 mL of deuterium oxide , vortexed for 5 min, and boiled for 30 min with occasional vortexing to avoid starch aggregation. These samples were freeze-dried again and re-dissolved in D2O (20 mg/mL) prior to 1H NMR analysis. The reference (δ = 0) used was 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, and 1H NMR spectra were collected at 80 °C.The ratios of α-1,4 and α-1,6 linkages in the GBE-treated normal corn starch samples were determined using a 400 MHz 13C NMR with cross polarization/magic angle sample spinning (CP/MAS NMR) experiments were conducted using an AVANCE 500  NMR instrument equipped with CP/MAS accessories at a frequency of 100 MHz under high-power decoupling conditions. Approximately 200 mg of starch was packed using a 4 mm-diameter rotor. Samples were spun at a rate of 5 kHz, with a spectral width of 3.1 kHz, acquisition time of 35 ms, and 2.2 k time domain points at 25 °C. At least 4000 scans were accumulated for each spectrum. The data processing and resonance peak spectrum integration were performed using the TOPSPIN 1.3 software . The double helix-to-amorphous ratio was obtained as described previously [High-resolution solid-state eviously .α radiation with a wavelength of two. The starch sample was scanned at a diffraction angle (2θ) ranging from 3° to 40° and a step time of 1 s. The relative crystallinity (RC) was calculated using the following equation, as previously described [Aa is the area of the amorphous region and Ac is the area of the crystalline region.X-ray diffraction (XRD) analysis was performed using an X-ray diffractometer  with a LYNXEYE XE detector (Bruker) at 40 kV, 40 mA, and a CuKescribed  using thg, 10 min), and the glucose content released at 20 min (G20) and 240 min (G240) was determined using a glucose assay kit. The total glucose (TG) content was determined by gelatinizing the starch, cooling it, adding 7 M KOH, and treating it with 20 μL amyloglucosidase. Free glucose (FG) was determined from a gelatinized starch sample in an acetate buffer solution. The values for RDS, SDS, and RS were calculated using the following formulas: RDS = (G20 − FG) × 0.9; SDS = ((G240 − FG) − (G20 − FG)) × 0.9; and RS = (TG − FG) × 0.9 − (RDS + SDS).The starch fractions were determined according to a previously described method  with slip < 0.05) in the SAS software .All experiments were performed at least three times. The data were reported as the means with standard deviation. Analysis of variance (ANOVA) was performed and differences in means of samples were analyzed using Duncan’s multiple range tests (PhGBE from P. horikoshii OT3 and CbGBE from C. bescii DSM 6725 were expressed in E. coli BL21(DE3) cells and purified by heat treatment followed by Ni-NTA chromatography. The PhGBE and CbGBE was purified 21.6- and 30.9-fold with a yield of 11.6% and 3.9%, respectively , and high-AM starch (~70% AM), were preferred, rather than AM, which contains only α-1,4 glycosidic linkages. lication . Thin-lae for AM .Thermus thermophilus HB8 and TkGBE in Thermococcus kodakarensis KOD1[CbGBE was found to produce significant quantities of maltohexaose and maltoheptaose after isoamylase treatment through TLC analysis , suggesting that the two GBEs successfully interacted with AM .CbGBE predominantly produced side chains with DPs of six and seven, consistent with the TLC pattern  made up the largest fraction and a decrease in the proportions of the B1, B2, and B3 chains was observed  increased slightly but that of the B1 chain was still predominant. The results of the detailed side chain length distribution on modified starches were dissimilar between the two GBEs. PhGBE showed the greatest change in DP 6−9 from modified starch, but DP 10−12 was dominant when AM was used as a substrate, as shown in the previous result. The findings indicate that the branching activity of PhGBE differs depending on the substrate.Further, the branching activity of observed . However1H NMR was used to analyze the ratio of α-1,6 linkages to the total glycosidic linkages in GBEs-modified starches. Native , control, and two GBE-treated starches showed two distinct peaks at 5.4 and 5.0 ppm, indicating the chemical shift of anomeric protons in α-1,4 and α-1,6 linkages, respectively. The percentage of each linkage was calculated from the integrals of the corresponding peaks in the spectra [PhGBE- and CbGBE-treated starch was 4.4% and 8.6%, respectively  between the two GBE-treated starches. Previous studies reported that α-1,6 linkages increased 7.1% and 8.2% after GBE treatment in waxy corn starch [ spectra . The abuectively . The finn starch .13C CP/MAS NMR, whereas the long-range molecular order (double-chain) of crystallinity was determined by XRD. CP/MAS resonance peaks are specific for amorphous sites in the 80–87 ppm and 103–104 ppm regions, whereas signals at 99–102 ppm are specific for the double helices [Starch crystallinity based on the ratio of double helices and amorphous (single-chain) sites, which is termed short-range molecular order, was determined by solid-state  helices ,21. The CbGBE-treated starches and 29.1:70.9 for PhGBE-treated starches. The degree of double helix of GBE-treated starches determined through 13C CP/MAS NMR analysis was lower than that of the native starch and higher than that of the control starch. 13C CP/MAS NMR examination also showed that the crystalline and amorphous regions of PhGBE- and CbGBE-treated starches did not differ significantly (p > 0.05).The double helix-to-amorphous site ratio for native corn starch was 42.1:57.9, consistent with that observed in a previous report . Controlθ), as reported previously [RC) was not possible using the TOPAS software. This indicated that GBEs-treated starches were unable to rebuild a crystalline structure  and crystallinity (XRD) was indicative of incomplete crystallinity and irregularly packed structures as well  and α-1,6-branch points, which were dependent on the nature of the GBE; the relative ratio of A chains and 1,6-branching points in CbGBE-treated starches was much higher than that in PhGBE-treated starches. CbGBE transferred relatively large quantities of shorter A chains , as well as overall A chains , to the acceptor chains to form branch points, whereas PhGBE formed slightly higher quantities of shorter A chains with DP 6–9, while longer A chains with DP 10–12 were formed in quantities similar to those of the control. CbGBE increased the SDS and RS contents, while PhGBE increased the RS content only. Therefore, we propose that the quantity of A chains and the DP distribution (DP 6−9 and DP 10−12) favors both RS and SDS formation (from CbGBE), whereas the quantity of shorter A chains (DP 6−9) is related to RS formation (from PhGBE). This suggests that A chains with DP 10–12 are related to the SDS content. This study also confirmed that a significant quantity of A chains (DP 6−12) and an increased number of branch points favor the formation of both SDS and RS fractions, slow the enzyme accessibility to starch and reduce the digestibility of starch.CbGBE (GH13) and PhGBE (GH57), showed different branching activities with the substrate they used. The branching activity of CbGBE contained both α-1,4 and α-1,6 linkages, whereas PhGBE showed limited activity for substrates containing α-1,4 linkages such as AM. Modified starch with GBEs showed PhGBE was remarkable with DP 6−9 and CbGBE was with DP 6−12 side chains. Subsequently, two types of GBE produced a different side chain distribution, degree of branch points, and nutritional fractions in starch. The CbGBE increased the amount of SDS and RS, but PhGBE only increased the amount of RS. The collective findings of this study indicate that GBEs could be effective for producing customized modified starch and control glucogenesis in vivo. Further, thermostable GBEs might be useful as a cost-effective strategy to reduce the processing time for the industrial production of starches with a low-GI value.Two thermophilic enzymes belonging to two different families,


The results of kidney transplantation are impacted by the categories of events responsible for patient death and graft failure. The objective of this study was to evaluate the causes of death and graft failure and outcomes after graft failure among kidney transplant recipients. A retrospective cohort study was conducted with 944 patients who underwent kidney transplantation. Outcomes were categorized in a managed and hierarchical manner.p<0.0001). As late causes of graft loss, death with the functioning kidney occurred earlier than recurrence and chronic rejection (p=0.008). The outcomes after graft failure were retransplantation in 26.1% and death in 21.4%, at a mean of 25.5 and 21.4 months, respectively. The crude mortality rate was 10.8% (n=102): in 35.3% cause of death was infection, in 30.4% cardiovascular disease, and in 15.7% neoplasia and in 6.8%, it was not possible to determine the cause of death. The rate of graft loss was 10.6%. The main causes of graft failure were chronic rejection (40%), acute rejection (18.3%), thrombosis (17.3%), and recurrence of primary disease (16.5%). Failures due to an acute rejection occurred earlier than those due to chronic rejection and recurrence ( It was possible to identify more than 90% of the events responsible for the deaths of transplanted patients, predominantly infectious and cardiovascular diseases. Among the causes of graft failure, chronic and acute rejections and recurrence were the main causes of graft failure which were followed more frequently by retransplantation than by death on dialysis. Renal transplantation is the treatment of choice for patients with advanced kidney disease, even when compared with more sophisticated dialysis modalities –4. In reIn addition, previous data have shown that extending the follow-up of transplanted patients beyond the time of occurrence of graft failure may reveal the negative impact of the loss of transplantation function on the survival of the previously transplanted patient . In the Thus, the objective of this study, carried out in a single Brazilian center, was to evaluate the distribution of the causes of death and graft failure, in a categorized way, through a managed and hierarchical system of information, as well as the incidence of clinical outcomes of patients who return to dialysis after kidney graft failure.This is a retrospective cohort study carried out with kidney transplant patients between 2002 and 2015, in the transplantation program of Hospital Israelita Albert Einstein , located in the city of São Paulo, Brazil. The city of São Paulo is the capital of São Paulo state and it is the most populous city and state in the country. The health service where the study was conducted is a private hospital that provides philanthropic services through a Ministry of Health funding program called PROADI-SUS. The project was approved by the local ethics committee.The initial data were extracted during the years 2016 and 2017 from an integrated hospital management system that performs the routine collection of health data from local programs. Thus, all patients included had the possibility of exposure to the risk for at least one consecutive year after transplantation. Patients of any age at the time of transplantation were eligible for this cohort, including transplants performed with pediatric recipients, all of whom had undergone kidney transplantation from a living or deceased donor in our service. The exclusion criteria were as follows: kidney transplant recipients combined with another solid organ  and transplants performed outside the PROADI-SUS program, that is, those who had supplementary or private medicine as a source of funding, since their follow-up after transplantation was performed independently of the program mentioned above.The demographic variables extracted were age, date of transplantation, sex, ethnicity, and donor type. The outcomes considered were graft loss, defined as the need for the permanent return to dialysis after transplantation, death, and loss of follow-up. The events of graft loss or death that occurred in the service itself were identified through the hospital management system. To identify the events that occurred in another health service or outside the health system (in the case of death), the outpatient returns of all patients were monitored by a nurse (author: PRB) responsible for information management (IMN). Through this monitoring, all patients were contacted by phone when they did not attend the consultations for more than 3 months between two consecutive visits. In cases of patients with less than 1 year of transplantation, the contacts were performed when the outpatient return period was not in accordance with the medical conduct indicated at the last visit or by the criterion of returns defined in the institution's care protocol, as described in Graft loss was classified in the following groups: primary nonfunction, when the graft lost occurred before function recovery; early death, when patients died after transplant surgery, but before function recovery; death with functioning graft; and graft failure. Deaths were classified into two groups: those that occurred after the patient had renal graft function  and those that occurred after the surgical procedure of the transplant, without the recipient having had renal graft function . The causes of death with the functioning kidney were categorized as infection, cardiovascular disease, neoplasia, or others. Cardiovascular disease was indicated as the cause of the event in both cases when it was the immediate cause of death and when it was the root cause. For the early complications, events that occurred in the 28 days following the transplant surgery were considered in the study, in those which a surgical or clinical complication was the root cause, among patients without graft function.Graft failure was categorized into thrombosis, acute rejection, chronic rejection, recurrence, or others. Regarding thrombosis, patients with arterial or venous renal thrombosis of the renal graft of mechanical cause were included in the study. All graftectomies of thrombosed grafts were evaluated by pathological anatomy. Those with evidence of an immune-mediated vascular injury (hyperacute or accelerated rejection) were categorized as acute rejection. In the chronic rejection category, cases of loss due to interstitial fibrosis and tubular atrophy, or with evidence of chronic cell-mediated or antibody-mediated rejection, and cases of transplant glomerulopathy were included . Cases wTo minimize the risk of classification bias, the IMN (author name: PRB) presented the event summary to the attending physician responsible for the clinical follow-up of the respective patient , and the physician indicated one of the categories described above. All categorizations indicated by the attending physicians were reviewed by a single supervising physician (LRM). In the case of disagreement, the records were reviewed, and when there was no consensus, the event was classified as being of unknown etiology.For the patients evaluated with graft failure, the identification of the outcome after the return to dialysis was performed by the IMN (author name: PRB) through follow-up of the evolution of the medical appointments in the program itself, while the patient maintained a follow-up with the medical team. After the loss of clinical follow-up in the transplantation program, the information was monitored through a phone call to the patient, guardian, dialysis clinic, and/or center for the notification, collection, and distribution of organs. Postfailure outcomes were classified as maintenance on dialysis, retransplantation, or death. In cases of death, a copy of the official death certificate was requested for date verification. Due to the inconsistency in completing the field of cause of death in the certificates of these patients, the cause of death was not considered for this analysis. p<0.05, considering a 95% confidence interval.All transplants in the period that met the eligibility criteria and did not present exclusion criteria were considered for the cohort; therefore, no sample number calculation was performed. The age variable was summarized as the mean and standard deviation because it presented a normal distribution, and for categorization, a multiple of 10 closest to the mean was used: 40 years. The follow-up measures had a nonnormal distribution, so they were summarized as medians, with the interquartile ranges  as a measure of dispersion. Categorical variables were summarized as percentages. The times to the occurrence of events were compared using the nonparametric Mann-Whitney test, and in the case of comparisons between three groups, an ANOVA analysis was used. Survival and cumulative incidence curves were calculated using the Kaplan-Meier method, and comparisons were made using the log-rank or Breslow-Wilcoxon test. Statistical analyses were performed using SPSS® 25 , and the graphs were constructed in GraphPad Prism 7 . Statistical significance was defined asBetween 2002 and 2015, 1,239 transplants were performed in our center. However, the following transplanted patients were excluded: 197 patients whose kidney transplants were combined with another solid organ , 93 transplants performed outside the program (with private or supplementary medicine as a funding source), and five patients who were transplanted through the program but completed follow-up outside the program. Thus, a total of 944 patients were included in the study .There was a predominance of male recipients  and of the white ethnicity , averaging 43.5±14.1 years, and 54.2% received grafts from deceased donors. At the time of transplantation, only 2.2% (n=21) of the patients were less than 18 years old, 38.9% (n=367) were between 18 and less than 40 years old, 53.4% (n=504) were between 40 and 65 years old, and 5.5% (n=52) were 65 years old or older. The follow-up time was 68.1 (range 31.6-112.4) months. In this period, 217 grafts were lost (23.0%): 92 were due to previously functioning graft failure (42.4%), 92 were due to death with the functioning kidney (42.4%), 23 were due to the primary nonfunction (10.6%), and 10 patients died due to surgical or early clinical complications without having presented renal function (4.6%). Among patients with a primary absence of function, three had acute tubular necrosis (ATN) without the recovery of function within 3 months of follow-up after transplantation (1.4%) and therefore remained on dialysis, and 20 had arterial or venous thrombosis of mechanical origin (9.2%). Staphylococcus aureus transmitted by the same donor, one due to a coagulation disorder, and another due to hemophagocytic syndrome. The distribution of the frequency of deaths according to the cause is summarized in p=0.47): infection at a mean of 44.1 (range 12.3-113.5) months, cardiovascular events at a mean of 47.4 (range 5.2-71.7) months, and neoplasia at a mean of 45.2 (range 24.8-109.6) months. The crude mortality rate was 10.8% (n=102), with a mean occurrence time of 35.2 (range 5.8-73.5) months after transplantation. One-, 5-, and 10-year patient survival were 96.6%, 91.5%, and 87.0%, respectively . InfectiThe rate of graft loss, censored for death, was 12.2% (n=115), with a mean occurrence time of 38.1 (range 2.3-84.9) months. So, at the end of 1, 5, and 10 years the death-censored graft survival was 95.8%, 91.4%, and 83.7%, respectively . Chronicp<0.001, compared with recurrence and chronic rejection), with 61.9% of the events occurring in the first year of transplantation. Loss due to recurrence occurred at a mean of 50.4 (range 20.0-86.5) months, with 21.0% of the events occurring in the first year, 37.1% between the first and fifth year, and 41.9% after the fifth year. Finally, chronic rejection loss was later than recurrence, occurring at a mean of 83.0 (range 42.9-105.8) months, but this difference was not significant (p=0.10). Only 34.9% of chronic rejection losses occurred within the first 5 years after transplantation. Disregarding surgical causes due to their immediate nature, loss due to acute rejection was the earliest occurrence, at a mean of 3.2 (range 0.76-32.3) months (p=0.008), with a mean occurrence after transplantation of 45.9 (range 14.8-81.8) months and with 22.8% of the events occurring in the first year, 21.8% between the first and third year, 18.5% between the third and fifth year, and 36.9% after the fifth year of transplantation.Excluding the losses and deaths in patients whose graft never had function , death with the functioning kidney was the main cause of loss , followed by chronic rejection , acute rejection , and recurrence . The other causes accounted for 3.3% of the losses (n=6). Death with a functioning kidney occurred earlier than recurrence and chronic rejection (p<0.001) and among those who received a transplantation under the age of 40 (p<0.001). There was a trend toward a better result among black recipients (p=0.07), and there was no difference regarding gender. Within 5 years of follow-up, no differences were observed according to the period in which the transplantation was performed.Graft survival uncensored for death at the end of 1, 5, and 10 years was 92.4%, 83.4%, and 72.7%, respectively. The status of the patients who lost the graft was followed up for a mean of 30.9 (range 13.0-53.4) months after returning to dialysis. Among these, 25 (21.7%) patients died, at a mean of 21.4 (range 10.4-46.5) months after returning to dialysis. One-, 3-, and 5-year patient survival after the return to dialysis were 92.4%, 81.3%, and 72.4%, respectively . Thirty According to the Brazilian Transplant Registry, in 2017 there were 185 active teams for kidney transplantation in the country, and 5,492 transplants were notified. Considering the vertical nature of the kidney transplantation program, since between 17% and 20% of all kidney transplants are performed in a single center, the average annual number of transplants performed at the center evaluated in the present study (between 60 and 70 transplants per year) was slightly above the national average. Therefore, it is considered that the long-term outcomes of a Brazilian, single-center cohort with average transplantation activity are presented here. Initially, we observed that patient and graft survival were comparable with results found in United States and Australian registry data, and deceased donor outcomes were slightly above the average of the state of São Paulo, where the study hospital is located, especially after 5 years of follow-up. We summarized the data used as a basis for comparison in We presented the frequencies of events responsible for deaths and graft losses, from a managed and hierarchical system, in addition to efforts to identify all the events that occurred outside the operation in the transplant center. It was observed that this strategy guaranteed the classification of 93% of the deaths and 99.1% of the losses. In an American study evaluating the outcomes of 1,317 consecutive kidney transplants, El-Zoghby et al. were unable to determine the cause of death in 31.2% of the patients evaluated (a percentage 4.6 times higher than ours), and the follow-up of the cohort was not available . In someThe pattern of distribution of causes of death with a similar frequency of events of infectious and cardiovascular origin differs from previously published Brazilian data , where tWhen analyzing the combined graft losses, we observed a similar number of grafts lost due to death in a functioning kidney receptor and renal failure in a previously functioning graft. At the beginning of the last decade, accurate data from the Australian records showed that death with the functioning kidney was one of the main causes of graft loss, however, with less impact than chronic graft loss, which was classified at that time as chronic allograft nephropathy , 29. MorAs expected, and similar to that previously observed, acute rejection was the cause of earlier graft loss, with more than 60% of events occurring within the first year. However, there was a small impact on the cohort (2.2%), reflecting the improvement in the quality of immunosuppression observed in the last decades and more sensitive techniques of pretransplant immunological evaluation –34. DespIn an exploratory way, we compared the graft survival results, uncensored for death, according to the variables that are managed in the program. As expected, the results were significantly better in living donor recipients and in those transplanted at a younger age. The best evolution is known to be in living donor recipients, and the reasons for this vast difference have been widely discussed, especially the shorter waiting time for transplantation and, therefore, fewer morbidities associated with the evolution of chronic kidney disease, better compatibility, and, mainly, the quality of the organ , 10, 11.The clinical outcomes of kidney transplantation do not end when the transplanted patient has the complete and irreversible failure of graft function. By prolonging the follow-up of patients beyond the period of graft failure, it is observed that the return to dialysis has a significant negative impact. The mortality of patients who lose graft function increases 2 to 3 times after the return to dialysis , 17, ranAnother variable with interference in the evolution of the patient after graft failure is the performance of a new transplant. When transplanted patients who return to dialysis are compared with those who have never been transplanted and are on the waiting list, it is observed that the risk of dying is three times higher among those who have already been transplanted . AlthougSome limitations should be highlighted in our study. Like any observational study, there is a threat to the validation of results, especially since we did not include detailed demographic information or details of the clinical evolution, such as the type of immunosuppression or details of morbidity. Despite the great effort to categorize the causes of death and loss, which generated a low index of unclassified causes of death, the chronic losses were categorized as chronic rejection, which occasioned a risk of classification bias. Finally, the study was limited in evaluating details about the follow-up of patients after graft failure. We have considered only the hard outcome such as death, retransplantation, or remaining on dialysis, without exploring more details or the variables associated with the chance of retransplantation and risk of death.In conclusion, this analysis has shown that the causes of loss and death are not very different from other data available in the literature, with one of the most relevant results being the demonstration that the active search for information in a managed and hierarchical way is able to clarify most of the causes of death and loss. In addition, this is the first Brazilian study to identify the outcomes of transplanted patients after graft failure. This information is of the utmost importance to the public health authorities involved in the planning of substitute renal therapies, given the increasing number of patients who return to dialysis after the failure of a previous renal graft and especially since, in the global context, Brazil has one of the largest public renal replacement therapy programs and the largest public transplant program.


Nano-biocomposites based on halloysite nanoclay and potato starch were elaborated by melt blending with different polyol plasticizers such as glycerol, sorbitol or a mixture of both. The effects of the type of plasticizer and clay content on potato starch/halloysite nano-biocomposites were studied. SEM analyses combined with ATR-FTIR results showed that a high content of sorbitol had a negative effect on the dispersion of the halloysite nanoclay in the starchy matrix. XRD results demonstrated that incorporation of halloysite nanoclay into glycerol-plasticized starch systems clearly led to the formation of a new crystalline structure. The addition of halloysite nanoclay improved the thermal stability and decreased the moisture absorption of the nano-biocomposites, whatever the type of plasticizer used. Halloysite addition led to more pronounced improvement in mechanical properties for glycerol plasticized system compared to nanocomposites based on sorbitol and glycerol/sorbitol systems with a 47% increase in tensile strength for glycerol-plasticized starch compared to 10.5% and 11% for sorbitol and glycerol/sorbitol systems, respectively. The use of a mixture of polyols was found to be a promising way to optimize the mechanical properties of these starch-based nanocomposites. These biomacromolecules are organized into a highly complex semi-crystalline structure that results from the biosynthesis of the starch granules by the plant [In recent decades, biopolymers  such as polysaccharides, and more generally bio-based polymers from renewable resources have attracted a great deal of attention from researchers and industry because of the increasing awareness of environment protection and the lack of certain specific fractions extracted from fossil reserves ,2. Amonghe plant ,6. Neat he plant . TPS shohe plant ,8,9,10. he plant ,12. Somehe plant ,14,15. Dhe plant  while prhe plant .2Si2O5(OH)4·nH2O, with n from 0 to 2, for hydrated and dehydrated halloysite, respectively. The tubular structure of halloysite results from the wrapping of the constitutive 1:1 clay layers under specific geological conditions. Typically, halloysite ranges in length from 300 nm to 1500 nm, with an external diameter of 40–120 nm and internal diameter of 15–100 nm [Up to now, researchers have focused on layered silicates, especially montmorillonites (MMT) . However5–100 nm . In cont5–100 nm . Consequ5–100 nm ,28. HoweThus, the aim of this work is to study the effect of the addition of halloysite into a starch-based matrix which is plasticized by glycerol, sorbitol or by a mixture of them, so as to reduce the water sensitivity and improve the thermal stability and mechanical strength. In particular, the effects of the type of plasticizer and nanoclay loading on the structure and properties of the obtained starch nano-biocomposites will be investigated using different characterization techniques such as scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared (ATR-FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), moisture content measurement and uniaxial tensile testing methods.®, a glycerol/sorbitol mixture (59/41 by weight) was kindly supplied by Roquette . The halloysite nanoclay with diameter of 30–70 nm and length of 1–3 µm (CAS 1332-58-7) was purchased from Sigma-Aldrich .Potato starch was supplied by Roquette  . The amylose and amylopectin contents were 20% and 80%, respectively. Glycerol was a 99.5% purity product . Sorbitol was kindly supplied by Tereos  with 98% purity. PolysorbThe nanocomposites were obtained from a two-step process with the first being the elaboration of a dry-blend (powder) by the mixing of starch, water and polyol. Such dry-blend protocol allows the subsequent preparation of plasticized starch with high plasticizer content without the exudation phenomenon, mainly thanks to the strong interactions established between the polysaccharide chains and the polyols . Then, tFirst, the starch/plasticizer dry-blends were prepared. In this process, native potato starch was dried overnight at 70 °C in a ventilated oven to remove the free water . Then, the dried starch powder was introduced into a Papenmeier turbo-mixer, and the plasticizer  was slowly added under mixing until a homogenous mixture was obtained. The mixture was then dried at 170 °C in a ventilated oven for 40 min and occasionally stirred, and then the dry-blend was recovered. To obtain adequate moisture content, a pre-determined quantity of water was added to the dry-blend after cooling and mixed in the turbo-mixer, resulting in a formulation containing 54 wt.% potato starch, 23 wt.% plasticizer and 23 wt.% water. Finally, the powder was stored in a polyethylene bag in a refrigerator at 6 °C overnight prior to processing.®) and Y for the weight percentage of halloysite nanoclay.To obtain nano-biocomposites, the plasticized starch powder was processed, with the addition of 3 wt.%, 5 wt.% and 7 wt.% of halloysite nanotubes compared to the weight of the dry-blend powder, in a counter-rotating internal batch mixer, Rheomix OS , at 70 °C for 20 min with rotor speed of 150 rpm. After melt mixing, the mixtures were then compression molded  at 110 °C, applying 18 MPa for 15 min. The obtained films (thickness approx. 1 mm) were then stored at 57% relative humidity at 23 °C for at least three weeks before characterization to obtain stabilized properties. The samples are designated as XY where X stands for the type of plasticizer . The film samples were mounted on a stub using double-sided adhesive tape and coated with a thin layer of gold (10–20 nm). The images were obtained at an operating voltage of 5 kV and ×10,000 magnification.−1 by accumulation of 32 scans at a resolution of 4 cm−1.IR spectra were recorded using a Thermo Scientific Nicolet iS10 FTIR spectrometer  in attenuated total reflectance (ATR) mode. IR spectra of films were obtained for wavenumbers ranging from 4000 to 650 cmα radiation (λ = 0.1542 nm) operating at 50 kV and 40 mA. The scanning region of the diffraction angle (2θ) was from 10° to 50° with a step size of 0.02°.X-ray diffraction analysis was carried out on a Bruker AXS D8 ADVANCE  using Cu-KThe thermogravimetric analysis was performed on a TGA Q5000 machine . Sample masses ranging from 2 mg to 3 mg were heated from 35 °C to 600 °C at the rate of 10 °C/min under air atmosphere (flow rate of 25 mL/min).Samples films were cut into square shapes. Then, the weight, length, width and thickness of the film were measured, allowing the calculation of the material density. Experiments were done in triplicate. The water contents of samples were estimated using a MB45 moisture analyzer  considering the weight loss measured after 1 h of drying at 105 °C.The uniaxial tensile strength, elongation at break and Young’s modulus were determined using a Universal Testing Machine MTS 2M . The samples were cut into a dumbbell shape and the tests were performed at room temperature with a constant deformation rate of 10 mm/min and a distance grip separation of 4 cm. Five specimens of each formulation were tested and the average values were calculated. Samples were conditioned at ambient temperature with relative humidity of 52 ± 2% for at least 3 weeks prior to testing.SEM images of plasticized starch and starch nano-biocomposites are displayed in Adding halloysite into glycerol plasticized starch resulted in a more uniform and continuous morphology of the starchy matrix. No aggregates were visible for nano-biocomposites based on glycerol plasticized starch B–D. EvenBesides, the presence of voids between the clay aggregates and the starchy matrix suggested a poor interface quality K,L. For ATR-FTIR was used to investigate the interactions between plasticized starch and halloysite nanoclay. The spectra of the native potato starch, halloysite nanoclay and plasticized starch nano-biocomposites containing 7 wt.% halloysite nanoclay are shown in −1 in the spectrum of native potato starch was attributed to the complex vibrational stretches associated with the free, inter- and intramolecular bound hydroxyl groups [2 into corn starch film [2 and starch. The same trend was observed for the nanocomposites plasticized with sorbitol, alone or mixed with glycerol.The broad band between 3000 and 3600 cml groups . Tang, erch film  with the−1 were related to the O–H stretching of inner-surface hydroxyl groups and inner hydroxyl groups, respectively. After addition into the glycerol plasticized matrix, the O–H stretching peak of inner-surface hydroxyl groups slightly shifted to a lower wavenumber, 3691 cm−1. Similar findings were previously observed by Schmitt et al. [−1, respectively. This suggested a decrease in the intermolecular interactions between the inner-surface hydroxyl groups of halloysite and the C–O–C groups of starch and/or plasticizers. Since the main difference between these nano-biocomposites was the nature of the plasticizer, it could be concluded that glycerol formed stronger and more stable hydrogen bonds with the inner-surface hydroxyl groups of halloysite compared with sorbitol. This explains why a better dispersion of nanofillers could be obtained in the case of the glycerol plasticized starch matrix.In the spectrum of halloysite nanoclay, the peaks at 3693 and 3623 cmt et al. . This shθ of 12.1°, 18.6°, 20.3°, 25.0° and 27.0° [θ of 15.4°, 17.4°, 19.9°, 22.5° and 24.2° which are characteristic of B-type starch crystalline structures [H-type and VH-type structures with characteristic peaks at 2θ of 17°, 19.9°, and 22.0°, respectively [H-type structure is linked to amylopectin recrystallization while the VH-type structure is attributed to amylose crystallization into single helical structure [Halloysite displayed the typical pattern of the dehydrated form with characteristic peaks at 2nd 27.0° . The patructures . After pectively ,36. The tructure . Importaθ of 12.1° and 27.0° remained visible in the patterns of the nano-biocomposites. The peak intensity increased with the halloysite content while the peak position remained almost unchanged with only a slight broadening of the peak at 2θ of 12.1°. In addition, the characteristic peaks of plasticized starch were also observed in the diffractogram of the nano-biocomposites, attesting that halloysite nanotubes were well dispersed in the starch matrix [H-type and VH-type crystallization peaks were observed for the starch/halloysite nano-biocomposites. The XRD patterns of nanocomposites based on glycerol displayed a new peak at 28.5°, compared with the pattern of unloaded plasticized matrix. Similar findings have been reported for starch/tunicin whiskers [After dispersion in the starch matrix, the halloysite characteristic peaks at 2h matrix . No signwhiskers  and starwhiskers . This wawhiskers , PA6/halwhiskers  and PVDFwhiskers . HoweverGenerally, the addition of nanoclays can generate two opposite effects on the thermal stability of polymer/clay nanocomposites: (i) a barrier effect which improves the thermal stability and (ii) a promoter effect which increases the thermal degradation process . An enha10%, T50% and T90% refer to temperatures at which weight loss is 10%, 50% and 90%, respectively, and Tmax corresponds to maximum degradation rate. The char residue (%) is the unburnt residue at 600 °C.The detailed TGA data for starch-based nanocomposites, and raw compounds are summarized in 10% and correlated to the sorbitol content. This behavior was attributed to the higher thermal stability of sorbitol compared to glycerol. For all starch-based nanocomposites containing 3 wt.% halloysite nanoclay, T50% was enhanced in comparison to the glycerol plasticized matrix, indicating that nanoclay had a barrier effect with an impact on degradation. This was attributed to the good dispersion of nanoclay in the matrix, even at low content. Considering the tube-like structure of halloysite nanoclay, an increase in the tortuosity was less obvious, thus the enhancement of thermal stability was more likely related to the strong interactions between the halloysite nanoclays and the matrix. Similar findings have been reported for polyethylene/halloysite [An increasing trend was observed for Tlloysite , siliconlloysite  and polylloysite .50% gradually increased with halloysite nanoclay content in glycerol plasticized nanocomposites. However, there was no significant difference in T50% as the nanoclay content increased from 3 wt.%, to 5 wt.% to 7 wt.% in nanocomposites of starch plasticized with sorbitol, alone or combined with glycerol. This finding suggests that some degradation promoter effect occurred and impacted the thermal stability with the addition of high levels of halloysite nanoclay. According to the SEM images, this could be related to the formation of aggregates in nanocomposites plasticized with sorbitol, alone or blended with glycerol, at high halloysite content (5 wt.% and 7 wt.%) which limited the barrier effect. Since the nanocomposites show different moisture content it is difficult to make a direct comparison of T50% values, especially for such multiple-step degradation. Then Tmax, which corresponds to the temperature at maximum degradation rate was determined from the DTG curves maximum. In this case, the overall gradual increase in thermal stability with the clay content is more obvious and seems particularly marked for 7 wt.%. For T90% and the char residue, whatever the type of plasticizer, both increased with the halloysite content due to the higher thermal stability of the halloysite nanoclays.TThe densities and moisture content of the different samples were determined after three weeks of equilibration, at room temperature and 52 ± 2 %RH. Results are summarized in It can be seen that the nanocomposites showed a slightly increased density when nanofiller content increased. This is due to the higher density of halloysite nanoclay. Since the density of glycerol is lower than that of sorbitol, nanocomposites based on glycerol exhibited lower density than nanocomposites based on sorbitol. For the same reasons, the densities of the nanocomposites with the mixed polyols system were in-between those of glycerol and sorbitol-based plasticized starch nanocomposites.glycerol is the water content of the nanocomposite with neat glycerol, Wsorbitol is the water content of the nanocomposite with neat sorbitol, Vglycerol and Vsorbitol are the respective nanocomposite volume fractions in the composites. The calculated values for sample P0, P3, P5 and P7 were 13.9%, 12.4%, 12.0% and 11.3% respectively. Interestingly, the experimental values were slightly lower but still in good agreement with the theoretical ones.Whatever the halloysite clay loading, the moisture content for nano-biocomposites with glycerol were higher than those recorded for nano-biocomposites with sorbitol alone or blended with glycerol. This is attributed to the higher hydrophilic character of glycerol compared to sorbitol . Besidesglycerol is the Young’s modulus of the glycerol-plasticized starch nanocomposite, Ysorbitol is the Young’s modulus of the sorbitol-plasticized starch nanocomposite, Vglycerol and Vsorbitol are the corresponding nanocomposite volume fractions in the systems. The ratio of Vglycerol/Vsorbitol is 0.6/0.4. Since the Young’s moduli of nanocomposites with sorbitol as plasticizer were much higher than those of nanocomposites with glycerol, the theoretical values were mainly dependent on sorbitol nanocomposites. The calculated values for P0, P3, P5 and P7 were 115.6, 127.6, 133.5 and 139.0 MPa, respectively. These values were lower than the experimental ones. This was probably due to (i) the much better dispersion state of nanofillers in the matrix based on the polyols mixture compared to that in the sorbitol-based matrix, and (ii) the lower experimental water content compared to the theoretical values [Young’s modulus, tensile strength and elongation at break of the different unfilled plasticized starch matrices and corresponding nano-biocomposites were measured. The main results are summarized in l values .glycerol is the tensile strength of the nanocomposite with glycerol-plasticized starch, Tsorbitol is the tensile strength of the nanocomposite with sorbitol-plasticized starch, Vglycerol and Vsorbitol are the corresponding nanocomposite volume fractions in the systems. The calculated values for P0, P3, P5 and P7 were 5.25, 5.68, 5.97 and 6.33 MPa, respectively. As expected, the experimental tensile strength values were higher than the theoretical ones.Similar to Young’s modulus evolutions, the tensile strength of plasticized starch was significantly impacted by the nature of the plasticizer. The tensile strength of starch plasticized with the polyols mixture was higher than the glycerol plasticized one. The highest tensile strength value was obtained with sorbitol. Such a trend was also recently observed in plasticized alginate obtained by thermo-mechanical mixing . This waThe nature of the plasticizer also impacted the elongation at break values of neat plasticized matrices. The elongation at break of the glycerol plasticized starch was lower than the sorbitol ones  probably due to the nanofiller dispersion state in the matrix. According to the morphological analyses, nanocomposites based on glycerol displayed good halloysite dispersion while nanocomposites based on sorbitol and the polyols mixture as plasticizers showed some aggregation of nanofiller at a high degree of loading. These results were in agreement with some previous observations off plasticized starch stored at high RH ,53. Due Plasticized starch/halloysite nano-biocomposites were successfully prepared. The influence of plasticizer type and filler loading on the microstructure and properties of the resulting materials were studied. Glycerol contributed to a more homogenous dispersion of halloysite nanotubes in the matrix compared to sorbitol, used alone or in combination, due to stronger and more stable hydrogen bonds between glycerol plasticized starch and halloysite, as revealed by ATR-FTIR analyses. The XRD results showed that incorporation of halloysite nanoclay led to the formation of a new crystalline structure attributed to amylopectin crystallization at the nanofiller interface which was mainly observed for nanocomposites based on glycerol or the mixture of glycerol and sorbitol as plasticizers. Whatever the type of plasticizer, the incorporation of halloysite increased the thermal stability and reduced the moisture content of the starch nano-biocomposites. The mechanical properties were also greatly impacted by the nature of the plasticizer. Glycerol-plasticized starch exhibited lower Young’s modulus, tensile strength and elongation at break than the sorbitol one. After addition of halloysite nanoclay, the improvement in tensile properties was more pronounced for glycerol plasticized systems due to the better dispersion state of halloysite compared to the systems based on sorbitol-plasticized starch. Finally, the use of mixtures of these polyols was found to be a good and promising way to optimize the mechanical properties of these starch-based nanocomposites, since the values of Young’s modulus and tensile strength were in-between those of glycerol- and sorbitol-based nanocomposites and higher than the theoretically expected ones.This work clearly showed that halloysite nanoclay is an effective and promising clay for enhancing the properties of plasticized starch. Nano-biocomposites based on starch/halloysite nanoclay represent an interesting alternative to replace some non-biodegradable materials in different fields such as packaging, agriculture or biomedical applications. For instance, in active packaging or in specific biomedical applications, halloysite nanoclay could be a good carrier for some active drugs due to its specific structure and surface properties. In future works, we will focus on developing different functional biomaterials based on starch/halloysite nanocomposites in order to broaden the properties and applications of starch-based materials.


The purpose of the investigation was to analyze fracture resistance and mode of failure of zirconium oxide (zirconia) abutments placed on dental implants bearing crowns of different esthetic materials: zirconia, lithium disilicate (LDS), and nano-ceramic resin, for replacing single teeth in the anterior sector. Eighty implant-abutment-crown units were divided into four groups: Group T-MC (control): 20 metal-ceramic crowns cemented onto titanium abutments; Group Z-Z: 20 zirconia crowns on zirconia abutments; Group Z-LD: 20 lithium disilicate crowns on zirconia abutments; and Group Z-NCR: 20 nano-ceramic resin crowns on zirconia abutments. Specimens underwent a fatiguing process (dynamic loading and thermocycling), followed by static loading to evaluate mechanical fracture resistance, and the mode of failure produced. Mean fracture resistance values were: Control Group T-MC, 575.85±120.01 N; Group Z-Z 459.64±66.52 N; Group Z-LD, 531.77±34.10 N; and Group Z-NCR, 587.05±59.27 N. In Group T-MC, fracture occurred in the prosthetic fixing screw in 100% of specimens. In Group Z-Z, 80% of fractures occurred in the fixing screw, 15% in the abutment, and 5% in the abutment and crown. In Group Z-LD, 60% of fractures were produced in the fixing screw and 40% in the abutment. In Group Z-NCR, 70% of fractures were produced in the fixing screw and 30% in the abutment. All the abutments and crowns analyzed have the potential to withstand the physiological occlusal forces to which they would be subject in the anterior region. Lithium disilicate and nano-ceramic resin crowns cemented onto zirconia abutments are a good restoration alternative for single implants in the anterior sector. A single missing tooth in the anterior sector is a complex challenge to the dentist, due to the need for restorations that are both esthetic and functional. One of the classic treatment options of choice is an implant-supported restoration with titanium abutment and metal-ceramic crown, which enjoys a high success rate of around 95% –3.Today, zirconia is considered a suitable material for prosthetic rehabilitation of anterior teeth due to its high biocompatibility, its esthetics (white color), and its mechanical properties, as it can withstand masticatory forces of over 150N –9.in vitro and in vivo trials, and for this reason, in recent years, zirconium oxide has been proposed as a material for fabricating this type of abutment [In vitro trials of zirconia’s fracture resistance have obtained widely varying results, with values ranging from 131 to 2517 N. Sailer [in vitro study was to analyze the fracture resistance and mode of failure produced (after fatiguing) of zirconia abutments bearing crowns fabricated from different esthetic materials: zirconia, lithium disilicate (LDS), and nano-ceramic resin, compared with the classic materials titanium abutments bearing metal-ceramic crowns used to replace a single missing anterior tooth.In response to the esthetic requirements of the anterior sector, ceramic materials have come into use for fabricating implant abutments. In 1993, Prestipino and Ingber designed and used an aluminum oxide abutment for the first time in a single-implant rehabilitation. But aluminum oxide has been found to present lower mechanical resistance to the occlusal forces exerted in the anterior sector in both abutment . Zirconi. Sailer  conducte. Sailer ,12. TherEighty tapered implants were used , 11.5 mm long with a diameter of 4.25 mm, with internal hexagonal prosthetic connections 2 mm in depth. The specimens were divided into four groups according to the crown and abutment materials:Group T-MC (control group) (N = 20 specimens) consisting of titanium prosthetic implant abutment screwed to the implant and a metal-ceramic crown cemented onto the abutment. The metal-ceramic crowns had Cr-Co cores fabricated using CAD-CAM technology  with a press veneering porcelain .Group Z-Z (N = 20 specimens): a zirconia abutment screwed to the implant, and a crown with a zirconia core and a press veneering porcelain  cemented onto the abutment.Group Z-LD (N = 20 specimens) consisting of a zirconia abutment screwed to the implant and a monolithic lithium disilicate ceramic crown screwed onto the abutment. The lithium disilicate crowns were designed and fabricated using CAD/CAM technology from pre-sintered blocks .Group Z-NCR (N = 20 specimens) consisting of a zirconia abutment screwed to the implant and a nano-ceramic resin crown cemented onto the abutment. The nano-ceramic resin crowns were designed and fabricated using CAD/CAM technology .All the abutments analyzed in the study were designed using CAD/CAM Echo2 software (Sweden&Martina) with standardized parameters. The total height of the abutment  was 10 mm. The abutment’s clinical height  was 8 mm. The abutment’s finish line diameter was 7mm . The angIn vitro fracture resistance evaluation followed the methods established in UNE-EN ISO 14801:2007. The implants were set in epoxy resin  with an elastic modulus of over 3 GPa, inside nylon tubes. To simulate peri-implant bone loss, 3 mm of the implant was left exposed from the implant collar in root-crown direction; the implants were set at an angle of 30° to the direction of loading .When all the implants had been set in tubes, the abutments were screwed onto the implants using a type IV titanium screw applying a torque of 30 N/cm with a prosthetic screwdriver equipped with a torque control device ISD900 . Access chimneys to the abutment screws were sealed with polytetrafluoroethylene (PTFE). Before cementation, the zirconia abutment, and the internal surfaces of the zirconia, lithium disilicate, and nano-ceramic resin crowns were prepared in various ways. All zirconia abutments were sand-blasted with 30μm silicon oxide particles (SiO2), at 2 bars pressure from a distance of 10 mm, using a Cojet Sand clinical sand-blaster (3M ESPE). These were then primed with silane . The zirconia crowns were also sand-blasted using the same technique as the abutments. The lithium disilicate crowns were etched with 9.6% hydrofluoric acid (Ultradent porcelain Etch) for 20 seconds and then, after washing and drying, brushed with silane . The nano-ceramic crowns were sand-blasted with 50 μm aluminum oxide particles at 2 bars pressure from a distance of 10 mm  applying silane afterwards. The titanium abutments did not receive any surface treatment before cementation. Metal-ceramic crowns were sand-blasted with aluminum oxide particles .The crowns were cemented  onto theAll specimens underwent an aging process consisting of dynamic loading and thermocycling. Dynamic loading was performed using a chewing simulator  and consisted of 240,000 masticatory cycles applying a vertical load of 8 kg with a vertical movement of 2.5 mm and horizontal movement of 2 mm below the crowns’ incisal edges. At the same time as dynamic loading, the specimens were subjected thermocycling , 1,548 thermal cycles per specimen, with changes in temperature from 5° to 55° every 30 seconds .After fatiguing and thermocycling, all specimens (crown-abutment) were tested by static load compression to evaluate their mechanical strength. A Shimadzu AG-100KN universal test machine (Shimadzu corporation) was used applying a cell load of 5000N; the cross head speed was 0.5 mm/min and the load was applied until fracture of the crown-abutment structure took place.When compression testing to the point of fracture had been performed, each specimen was examined under an optical microscope  to analyze the type of fracture and its location, classifying the fractures as follows: crown fracture without abutment fracture; abutment fracture without crown fracture; fracture of both crown and abutment; fixing screw fracture.All the data obtained were processed with TRAPEZIUM-X software . Descriptive statistics were calculated for the force variable: mean, standard deviation, minimum, maximum, and median. Confidence intervals of 95% were computed for mean values. A one-way ANOVA general linear model (GLM) was developed to determine if the mean level of fracture resistance could be considered homogeneous or not between groups. The significance level used in analysis was 5% (α = 0.05). Multiple comparisons were made using the Bonferroni test.During the application of dynamic load cycles and thermocycling, none of the specimens suffered fractures, delamination, or screw loosening. In this way, all specimens were available for fracture resistance evaluation .Fracture resistance evaluation obtained the following mean values: For group T-MC, mean resistance was 575.85±120.01 N; Group Z-Z 459.64±66.52 N; group Z-LD 531.77±34.10 N; and for Group Z-NCR mean fracture resistance was 587.05±59.27 N .One-way parametric ANOVA tests were used to determine homogeneity and statistic significant differences in resistance between the groups . The BonWhen the type of fracture and fracture location were analyzed, the following overall distribution was observed: 77.5% of all fractures were in the fixing screw between implant and abutment (62 fractures), 21.3% were located in the abutment but this only occurred among specimens with zirconia abutments (17 fractures), and 1.3% were located simultaneously in the crown and the abutment (1 fracture). In the T-CM control group, no fractures occurred in the abutment; all specimens (100%) suffered fractures in the fixing screw .In the Z-Z Group, most fractures were produced in the screw (80%), although in three specimens out of 20 (15%) fracture was located in the abutment; and in one case (5%), both the abutment and crown fractured. In the Z-LD Group most fractures were located in the fixing screw (60%) and the rest in the abutment (40%) .2 = 10.614).In the Z-NCR Group, most specimens suffered fracture in the screw (70%) , and theThe present study analyses the fracture resistance of zirconia abutments on implants, as well as the type of fracture produced. The zirconium dioxide abutments were restored with crowns fabricated from different materials, comparing these with the classic combination of titanium abutment bearing a metal-ceramic crown (Control Group), intended to replace a single missing tooth in the anterior sector. The study design followed norms established in UNE-EN ISO 14801:2007 for trials of endosseous dental implants subjected to dynamic fatiguing. The procedures followed have been used in many other published studies ,13,14.in vitro studies [To reproduce the conditions that restorations are subject to in the oral medium  specimens underwent an artificial ageing process consisting of dynamic loading and thermocycling. The literature includes various  studies ,15–17 th studies .7 (475±252N), but Gehrke [in vitro trial by Martínez-Rus et al. compared the fracture resistance of zirconia abutments supporting monolithic lithium disilicate crowns, lithium disilcate crowns with a manually-applied esthetic covering, and zirconia crowns, all reproducing the anatomy of the upper central incisors. The results showed that the zirconia abutments supporting both types of disilicate crown obtained slightly higher results (363–392 N) than samples restored with zirconia crowns (340 N) [Analyzing the fracture resistance data obtained in the study, the control group specimens with titanium abutment and metal-ceramic crown (T-MC Group) obtained mean values of 575.85±120.01N, which are similar to values obtained by Protopapadaki   .Lastly, the zirconia abutment with nano-ceramic resin crown group (Z-NCR Group), obtained a fracture resistance value of 587.05±59.27 N, which cannot be compared with any other research, as no other literature has dealt with this variable for zirconia abutments and crowns of this hybrid material. Authors such as Ferrario et al. , have foBased on these results, we can recommend the use of zirconia abutments in the anterior sector, especially in cases where, due to lack of bone, the implant has been positioned inclined to vestibular. In these case it is important to choose the material not only by their mechanical properties, but aesthetic ones. Thin thickness of the gingiva at the cervical level, in these situations, can make the material positioned under the gum visible. Zirconia is a white material, so it masks better than titanium under the gum, and also provides high strength values for anterior sector. The use of this type of abutment in the posterior sector would be extremely risky given the mechanical requirements in this area.The types of fracture registered were as follows: in the titanium abutment and metal-ceramic Crown group (T-MC Control Group), fractures occurred in the prosthetic fixing screw in all samples, a finding that coincides with Sghaireen , who obtThe majority of the studied samples fractured at the level of the abutment-implant union screw, so the results obtained from the fracture analysis can not be argued with the parameters studied in this article . The fracture of the screw depends on other variables such as screw material, type and height of implant connection, screw insertion torque and others.The main limitation of our study was its own nature, be an in vitro study. Although attempts have been made to reproduce the conditions of the oral environment, the results can not be extrapolated to 100% at the clinical level. We think that this research should be continued with a higher level of evidence by conducting a clinical trial.in vitro study have the potential to withstand the physiological occlusal forces that occur in the anterior region. The group presenting the lowest fracture resistance was the group of zirconia abutments bearing zirconia crowns (Group Z-Z) with statistically significant differences in comparison with the other groups. Lithium disilicate crowns and nano-ceramic resin crowns cemented onto zirconia abutments are a good option for implant-supported single restorations in the anterior sector. The weakest part of the implant-abutment-crown complex in all groups was the fixing screw.Within the limitations of this study, it may be concluded that: All the abutments and crowns fabricated from the materials evaluated in the present