Cancer Med. 2020; 9: 1515–1528. https://doi.org/10.1002/cam4.2815.Gao Y, Wang F, Zhang L, et al. LINC00311 promotes cancer stem‐like properties by targeting miR‐330‐5p/TLR4 pathway in human papillary thyroid cancer. wileyonlinelibrary.com), has been retracted by the journal's Editor‐in‐Chief, Dr Stephen Tait, and John Wiley & Sons Ltd. The retraction has been agreed due to concerns regarding image manipulation, which were brought to the journal's attention by a third party. A detailed investigation confirmed substantial inconsistencies. As a result, the journal no longer has confidence in the results and conclusions reported.The above article, published online on 02 January 2020 in Wiley Online Library ( Cancer Med. 2020; 9: 1515–1528. https://doi.org/10.1002/cam4.2815.Gao Y, Wang F, Zhang L, et al. LINC00311 promotes cancer stem‐like properties by targeting miR‐330‐5p/TLR4 pathway in human papillary thyroid cancer. wileyonlinelibrary.com), has been retracted by the journal's Editor‐in‐Chief, Dr Stephen Tait, and John Wiley & Sons Ltd. The retraction has been agreed due to concerns regarding image manipulation, which were brought to the journal's attention by a third party. A detailed investigation confirmed substantial inconsistencies. As a result, the journal no longer has confidence in the results and conclusions reported.The above article, published online on 02 January 2020 in Wiley Online Library (


Finding new agents to enhance the strength of alkali-activated (ground granulated blast furnace slag) GGBS is beneficial for environmental protection. Here, we reveal the effect of pregelatinized starch-modifying calcium silicate hydrate (C-S-H) particles on the nanostructure tailoring of NaOH-activated GGBS hydrates. The results show that, for the synthetic modified C-S-H, the pregelatinized starch absorbs on the surface of C-S-H, which modifies the silicate chains and crystal structure. Adding pregelatinized starch-modified C-S-H particles can tailor the chemical structure of calcium silicaluminate hydrate (C-A-S-H) formed from GGBS hydration by increasing the mean chain length (MCL) and decreasing the Al/Si ratios. When adding C-S-H particles modified by 0.1% pregelatinized starch, the MCL of C-A-S-H is increased by 344.5% and the Al/Si ratio is decreased by 16.0%. The compressive strength of NaOH-activated GGBS samples can be enhanced by adding pregelatinized starch-modified C-S-H particles, while the addition for modified C-S-H does not significantly affect the flexural strength. The high strength of hardened blocks of hydrated GGBS is related to the long MCL silicate chains. These findings provide a potential application of pregelatinized starch-modifying C-S-H particle acting as strength-enhancing agents. Goduction . Recent oduction ,8 and isoduction , anti-saoduction  and carboduction ,11,12. Goduction ,14,15. F2O3 and SiO2 are the main compositions [3+ for Si4+ occurs at bridging positions, a charge imbalance is generated. This charge imbalance is compensated by positive ions such as Na+ ions, Mg2+ ions and Ca2+ ions in gels. The N-C-S-H forms by the sodium ions balancing the charges of silicates chains. The properties of GGBS are directly related to the nature and structure of C-A-S-H gels. Compared to C-S-H chains in Portland cement, the chains in GGBS hydrates are longer, since aluminum-oxide tetrahedra make up silicate chains defects in C-S-H [29Si in silicates depends on the most recent coordination number Qn, where n  is the number of oxygen atoms shared by each tetrahedron with adjacent tetrahedra [n distribution of silicates chains in GGBS hydrates. For example, the waterglass induces the C-A-S-H gels with high Q2, Q3 or QPoly contents [2 unit content [2CO3 salts generate C-A-S-H gels dominated by Q1 and Q2 units [From the perspective of the GGBS hardening mechanism, the activator can generate calcium silicate hydrate (C-S-H), which is the source of activated GGBS strength. Up to now, the discovered C-S-H varies with about 40 types in cementitious materials. The nanostructure of classic C-S-H was considered to be the aggregates of tobermorite-like nanocrystals, jennite-like nanocrystals and disordered gels. This nanostructure was easily distorted by numerous factors such as calcium to silicon (Ca/Si) ratio, water to cement (w/t) ratio and impurity ions . In GGBS the alkaline oxides CaO and MgO, and acidic oxides Alositions ,16, whicositions ,18,19,20ositions ,22. Compin C-S-H . In cemetrahedra ,24,25. Dcontents ,27, and  content . AnotherQ2 units . In addin chains. However, research on C-A-S-H chain tailoring in GGBS hydrates is still lacking. Since the properties of materials depend on their structure, the method proposed here tailors the micro–nano structure through introducing synthetic C-S-H into activated GGBS. Considering the source of activated GGBS is relative to C-A-S-H generation, the addition of synthetic C-S-H in the hydration of GGBS has the potential to enhance the early strength of alkali-activated GGBS.Recent references show that different activators can cause the nanostructure of GGBS hydrates to be dominated by certain QThrough the use of bottom-up nanotechnology, the nanostructure of C-S-H has been modified and the healing of defects has been critical to the improvement in performance. The synthesis method of “polymer-inserting C-S-H”  and “poln distribution firstly. And then, the chemical structure and morphology of the three classic C-S-H particles were confirmed by XRD, FTIR, NMR and SEM. After this, three classic C-S-H particles were introduced to the NaOH-activated GGBS materials. The mechanism of microstructure tailoring of GGBS hydrates were revealed. FTIR and NMR were used to obtained the distribution changes of the Qn distribution in silicate chains. To verify the effect of synthetic C-S-H, the mechanical properties were also evaluated. This research provides a cheap and environmentally friendly method to enhance activated GGBS materials.Inspired by this, this paper synthesized three classic pregelatinized corn-starch modifying C-S-H particles with different Q2SiO4·9H2O; Sigma-Aldrich , ≥98%; 1.81 g powder dissolved in 50 g water), pregelatinized starch solution (dissolved in 100 g water) and hydrated calcium nitrate (Ca(NO3)2·4H2O; Sigma-Aldrich, 99%; 2.18 g powder dissolved in 50 g water). An amount of 2 g NaOH solid powders was added to adjust the pH to 12.2. The Ca/Si molar ratio of the synthetic pregelatinized starch-modifying C-S-H samples was 1.0. In the mixing process, solution A was prepared by first mixing the pregelatinized starch solution with the Na2SiO4·9H2O solution. The Ca(NO3)2·4H2O solution was then added to solution A with stirring and a white precipitate formed immediately. The sample was hardened at 60 °C for 24 h with stirring. Before characterization, the samples were washed twice with 1000 mL deionized water and then dried at 40 °C. The addition of pregelatinized maize starch was 0.0%, 0.1% and 0.3% by mass of solids (the sum of Na2SiO4·9H2O and Ca(NO3)2·4H2O powders), which was designated C-S-H, STACSH01 and STACSH03, respectively. The binding property of pregelatinized maize starch is relative to the hydrogen bonding sites (hydroxyl hydrogen and oxygen). The main chemical composition of GGBS is shown in The C-S-H samples were synthesized by mixing aqueous hydrated sodium silicate solutions  spectra were recorded using a 4 mm probe at a spin rate of 15 kHz at room temperature. Si CPMAS experiments were performed with a delay time of 3 s and a contact time of 1 ms using 5000 scans. Deconvolution was used to assign resonances to individual species using a combination of Gaussian and Lorentzian functions. The micromorphology of the samples was observed using SEM . The porosity and pore structure of GGBS mortars after 3 days of curing were characterized by the MIP method using AutoPore Iv 9510 . The mean chain length (MCL) and the Al/Si ratio were calculated using Equations (1) and (2), respectively [XRD data were obtained using a Rigaku-D/max 2550VB3 + X-ray diffractometer with graphite-monochromated Cu Kα radiation λ = 1.541 Å) generated at 40 kV and 100 mA. Scanning was performed with 2θ in the range of 5° to 70° and a scanning speed of 5°/min. The infrared absorption spectra of the samples were analyzed using FTIR  using the KBr pellet method.  Å genera−1 due to Si-O-Si bending vibrations. The band at 974 cm−1 is assigned to the Si-O stretching vibrations of Q2 tetrahedra. The bands at around 1640 cm−1 and 3460 cm−1 belong to stretching vibrations of -OH groups in H2O or hydroxyls. The group of bands near 447 cm−1 belong to the deformation of silicate tetrahedra. In the STACSH01 and STACSH03 samples, the 2932 cm−1 and 2936 cm−1 bands correspond to methyl groups (-CH3) [3 bonds increases gradually. There are no new chemical bonds of Si-O-R (R represent other groups) existing in the STACSH01 and STACSH03 samples, which demonstrates that pregelatinized starch may absorb on the surface of nanoscale C-S-H units. Other evidence for this absorption is the shifting of -OH bands at 1643 cm−1, 3462 cm−1 and 3458 cm−1 which is due to hydrogen bonding. Krautwurst [s (-CH3) ,39,42. Iautwurst  describe29Si NMR spectra of C-S-H. 29Si NMR spectra of STACSH01. 29Si NMR spectra of STACSH03. Peak fitting is performed before and after the pregelatinized starch modification to obtain the Qn distribution of silicate chains. The chemical shifts of −78.0 to −82.5 ppm belongs to Q1, −84.0 to −89.0 ppm belongs to Q2 and −89.0 to −104 ppm belongs to Q3 [2, the major peak at around −84.5 ppm and overlapped peak at around −89.5 ppm, were found in all samples. 2 is 45.3% + 50.4% = 95.7% and the content of Q3 is 4.3%. For STACSH01 samples, the content of Q2 is 39.6% + 47.9% = 87.5%, and the content of Q1 and Q3 is 3.1% and 9.5%, respectively. For the STACSH03 samples, the content of Q2 is 39.2% + 50.7% = 89.9%, and the content of Q1 and Q3 is 5.1% and 4.4%, respectively. Compared with C-S-H, the addition of 0.1% pregelatinized starch increases the content of Q3 silicate chains by 120.9% in STACSH01 samples and, when the addition of pregelatinized starch is 0.3% (STACSH03 samples), the content of the Q3 tetrahedron decreases to be similar to C-S-H. In addition, the content of the Q1 tetrahedron increases with the addition of pregelatinized starch. This indicates that the pregelatinized starch content affects and rearranges the nanostructure of C-S-H silicate chains. Theoretically, the C-S-H with a high content of Q3 tetrahedra has a better performance than that with a low content of Q3 tetrahedra. Yang Zhou [3 enhanced the Young’s modulus according to the nanoindentation measurement by destroying the layered structure. The XRD results show that the layered pregelatinized starch/C-S-H particle structure was distorted. However, in our research the STACSH03 samples have the same content of Q3 as the C-S-H sample, which indicates that layered structure changes are not due to Q3 formation but the absorption of pregelatinized starch. J.J. Beaudoin [3 signals by forming polymer–Q2 groups. If the pregelatinized starch bonds with C-S-H via Si-O-R, the Q3 content increases with decreasing of Q2. This explanation does not match the NMR analysis of the STACSH03 sample nanostructure. Thus, the speculation about pregelatinized starch absorbing on the surface of C-S-H and then modifying the nanostructure of silicate chains matches the results of XRD, FTIR and NMR.gs to Q3 ,24,25,33−1 and 964 cm−1 belong to Q2 tetrahedra. The bands at around 1640 cm−1 and 3480 cm−1 belongs to -OH groups in H2O or hydroxyls. It is noteworthy that bands at about 520 cm−1, 800 cm−1 and 1023 cm−1, which represent Al-O-Al, Si-O-Al and Si-O-Al bonds in C-(A)-S-H, respectively [−1 and 1023 cm−1 signals was due to their low content, and the supplementary information was obtained by NMR below. The FTIR results demonstrated silicate chain tailoring of GGBS by addition of pregelatinized starch-modified C-S-H particles. Since the performance is crucial relative to the material’s nanostructure, the C-A-S-H nanostructure change dominates the strength of GGBS at the molecular level. In order to obtained the detail of silicate chains, a NMR spectrometry analysis was conducted below.ectively , appeare0 (−68.0 to −76.0 ppm), Q1 (−78.0 to −82.0 ppm), Q2(0Al) (around −83.5 ppm), Q2(1Al) (around −85.5 ppm), Q3(0Al) (around −90.0 ppm) and Q3(1Al) (around −97.5 ppm) [0, Q1, Q2 and Q3 content is 10.4%, 4.1%, 7.1% + 29.1% = 36.1% and 46.0% + 3.3% = 49.3%, respectively. In 0 contents are 2.9%, 0.5% and 3.1%, the corresponding Q1 contents are 2.3%, 1.0% and 3.0%, the corresponding Q2 contents are 40.2%, 34.4% and 33.4%, and the corresponding Q3 contents are 54.4%, 64.1% and 60.4%. The synthetic C-S-H particles affect the Qn distribution of C-A-S-H generated in the NaOH-activated GGBS systems. The addition of PCSH0, CSH01 and CSH03 decreases the content of Q0 species, which belongs to the anhydrous GGBS. This indicates synthetic C-S-H particles enhance the NaOH activation process. Another finding is that, compared to the G0 sample, the content of Q2(1Al) also decreases in the PCSH0, CSH01 and CSH03 samples, which demonstrates that the replacement of Al3+ for Si4+ can be tailored by adding pregelatinized starch-modified C-S-H particles. To determinate the detailed nanostructure of silicate chains of hydrates, the MCL value and the Al/Si ratio were evaluated. In 7.5 ppm) ,44. In F0) is dissolved in an alkaline solution. At that time, the early precursors formed by the polymerization of silicate chains are the main source of strength of GGBS. The application of modified C-S-H to GGBS resulted in a significant reduction in the Q0 content of CSH01 and CSH03 during the experiment, probably due to the heteronucleation process causing Q0 chains to accumulate on the different active sites of modified C-S-H with a different structure. We speculate that the pregelatinized starch-modified C-S-H particles induce the formation of the C-A-S-H precursor with a specific structure and the subsequent reaction is controlled by a sufficient number of precursors, which determines the final chemical structure of C-A-S-H. This process appears to affect the C-A-S-H structure by influencing the Al/Si ratio, which can be verified by the NMR analysis. In summary, the addition of modified C-S-H particles affects the precipitation process of the polymerized silico-oxygen tetrahedra. And the precursor will first nucleate and grow at the active site of the externally modified C-S-H. Since the microstructural change can theoretically enhance the macroscopic mechanical properties, optimizing the mechanical properties can be achieved by increasing the MCL value, which is verified below. The MCL of C-A-S-H increased due to the structural adaptation of these active sites, which represents a promising new approach to GGBS activation.According to the recent finding ,45, C-S-n distribution of silicate chains. A similar phenomenon can be seen in nanosilica-enhanced GGBS hardened blocks, where nanosilica increases the 28 d compressive strength by 8.3–19.22% [3–19.22% . The nanTo enhance the understanding of basic mechanisms in tailoring nanostructures of activated GGBS system with lower NaOH usage, pregelatinized starch-modified C-S-H particles were synthesized, firstly, and then introduced into the GGBS hydration process. The nanostructures of both pregelatinized starch-modifying C-S-H and C-A-S-H generated from GGBS hydration were revealed. And the potential application in strength enhancement was also evaluated.Three synthesized C-S-H with different nanostructures can be obtained by the addition of pregelatinized starch. The pregelatinized starch absorbs on the surface of C-S-H and changes the silicate chains and crystal structure. The chemical structure of generated C-A-S-H from GGBS hydration can be tailored by pregelatinized starch-modified C-S-H particles. When adding C-S-H particles modified by 0.1% pregelatinized starch, the MCL is increased by 344.5% and the Al/Si ratios decreased by 16.0%. Compared to the pure NaOH-activated GGBS sample, the compressive strength of samples with added C-S-H particles modified by 0.1% pregelatinized starch was improved by 9.5% for 3 d, 16.6% for 14 d and 12.7% for 28 d. And the long MCL silicate chain may cause high strength in hardened blocks of hydrated GGBS. The potential application of pregelatinized starch-modifying C-S-H particles as strength-enhancing agents can be confirmed.


A disintegrin and metalloprotease with thrombospondin motif (ADAMTS) 4 might serve as a therapeutic target in treatment of heart failure and cardiac fibrosis.Heart failure is a condition with high mortality rates, and there is a lack of therapies that directly target maladaptive changes in the extracellular matrix (ECM), such as fibrosis. We investigated whether the ECM enzyme known as The effects of pharmacological ADAMTS4 inhibition on cardiac function and fibrosis were examined in rats exposed to cardiac pressure overload. Disease mechanisms affected by the treatment were identified based on changes in the myocardial transcriptome. Following aortic banding, rats receiving an ADAMTS inhibitor, with high inhibitory capacity for ADAMTS4, showed substantially better cardiac function than vehicle-treated rats, including ∼30% reduction in E/e′ and left atrial diameter, indicating an improvement in diastolic function. ADAMTS inhibition also resulted in a marked reduction in myocardial collagen content and a down-regulation of transforming growth factor (TGF)-β target genes. The mechanism for the beneficial effects of ADAMTS inhibition was further studied in cultured human cardiac fibroblasts producing mature ECM. ADAMTS4 caused a 50% increase in the TGF-β levels in the medium. Simultaneously, ADAMTS4 elicited a not previously known cleavage of TGF-β-binding proteins, i.e. latent-binding protein of TGF-β and extra domain A-fibronectin. These effects were abolished by the ADAMTS inhibitor. In failing human hearts, we observed a marked increase in ADAMTS4 expression and cleavage activity.Inhibition of ADAMTS4 improves cardiac function and reduces collagen accumulation in rats with cardiac pressure overload, possibly through a not previously known cleavage of molecules that control TGF-β availability. Targeting ADAMTS4 may serve as a novel strategy in heart failure treatment, in particular, in heart failure with fibrosis and diastolic dysfunction.  Cardiac fibrosis attenuates cardiac function and accelerates the development of heart failure and is associated with poor outcomes.4–6 The unmet need for therapies that target the cardiac ECM has been highlighted by the scientific community8 and leading medical societies.10Heart failure is a major public health problem due to its increasing prevalence and high mortality rates.11 Secreted metalloproteases such as A disintegrin and metalloprotease with thrombospondin motif (ADAMTS) are potential contributors to cardiac ECM remodelling via their preference for cleaving glycosylated proteins.12 ADAMTS4 is expressed by fibroblasts in the healthy human13 and failing murine heart14 and is up-regulated in the fibrotic injured heart15 and cardiac cells in response to inflammatory stimuli.16 We have previously demonstrated beneficial effects of reducing ADAMTS-mediated versicanase activity in the pressure-overloaded heart.16 However, the effects of more selective targeting of ADAMTS enzymes, and the mechanism for a potential beneficial effect of ADAMTS4 inhibition in cardiac disease, have not been explored.Maladaptive remodelling of the ECM is driven by altered proteolysis and synthesis of its components.17 to determine the therapeutic potential and main molecular effects of ADAMTS4 inhibition in pressure-overloaded rat hearts. We demonstrate that pressure-overloaded rat hearts treated with the ADAMTS inhibitor are protected against fibrosis and cardiac dysfunction, and we reveal a novel role for ADAMTS4 in the mobilisation of latent transforming growth factor (TGF)-β through cleavage of extracellular domain A (EDA)-fibronectin. The translational potential of ADAMTS4 as a therapeutic target is supported by the findings of the beneficial effects of ADAMTS4 inhibition in rat hearts following pressure overload and that substantially increased ADAMTS4 activity is observed in human heart failure.We hypothesized that ADAMTS4 is a therapeutic target in cardiac dysfunction and fibrosis, due to its effects on ECM components. In the present study, we have used a small molecule that inhibits ADAMTS enzymes with a high potency for ADAMTS4 inhibition2.2.1online, Table S1).The effect of the ADAMTS inhibitor on cardiac function in the pressure-overloaded heart was examined in rats that were subjected to aortic banding (AB) or to sham operation. In total, 24 animals were included in each AB group, whereas six rats were included in each sham group. To ensure an even distribution of the degree of stenosis in the AB groups, the rats were stratified and randomized to the treatments according to echocardiographic measures that were taken on Day 1 after surgery  that underwent AB using an o-ring around the ascending aorta, as described.19 Sham rats that received a loose suture around the aorta (ADAMTS inhibitor treatment study) or received surgery without the insertion of o-ring  served as controls. During surgery, rats were intubated and fully sedated by ventilation with a mixture of 98% oxygen and 2% isoflurane on a Zoovent . Subcutaneous administration of 0.1 mg/kg buprenorphine was given as pre- and post-operative analgesia. All experiments were performed in accordance with the Norwegian Animal Welfare Act and the National Institutes of Health guidelines20 that conform to European Parliament Directive,21 and after approval from the Norwegian Animal Research Authority (FOTS ID 7737 and FOTS ID 20208).For the ADAMTS inhibitor treatment study, male Wistar rats  underwent AB through placement of a suture around the ascending aorta, as previously described.2.3Conflict of interest).17 Pharmacokinetic studies and simulations demonstrated that a dose of 15 mg/kg/day of the ADAMTS inhibitor yielded an unbound plasma concentration in the steady state of 270 nM , anticipated to lead to a similar organ level.22 Based on a previously reported IC50 of 26 nM for ADAMTS4 and 860 nM for ADAMTS5,17 the estimated plasma concentration was 10 times the reported half-maximal inhibitory concentration (IC50) for ADAMTS4, which indicated sufficient efficacy in the case of ADAMTS4, while constituting 30% of the IC50 for ADAMTS5 and 1% of the IC50 for matrix metalloproteinase-2.17 A microsuspension of the ADAMTS inhibitor or vehicle was administered by oral gavage once daily starting from Day 3 post-AB.The hydroxamate-based small molecule designated ‘13n’, here called ‘ADAMTS inhibitor’, was generously provided free of charge by AstraZeneca  under anaesthesia with 1.5–2% isoflurane. Body temperature was kept around 37°C by warm air. Cine-MRI was acquired in the true short-axis and the four-chamber long-axis orientation in an electrocardiogram and respiratory gated manner. The rats were anaesthetized with 5% isoflurane before being euthanized by cardiac exsanguination. After the left ventricle and septum had been separated from the right ventricle, the cardiac ventricles and lungs were weighed and stored for later analyses.We performed echocardiography and magnetic resonance imaging (MRI) 8 weeks after surgery to assess cardiac structure and function, as has been described previously.2.5q-values) <0.15 was used to define the transcripts as differentially expressed genes (DEGs). The list of DEGs was annotated through the use of gene ontology (GO) enrichment analysis using the statistical overrepresentation test in g:Profiler (https://biit.cs.ut.ee/gprofiler/gost) and analysed through the use of Ingenuity pathways analysis (IPA)  to identify potential upstream regulators.Collagen content was determined by the use of high performance liquid chromatography (HPLC), while the amounts of fibronectin, latent TGF-β-binding protein (LTBP) 1, and versican DPEAAE-immunoreactive fragments were determined by immunoblotting of ECM-rich fractions. The RNA was isolated and analysed by RT–qPCR to determine the expression of genes that encode ADAMTS enzymes that cleave proteoglycans, and genes associated with heart failure mechanisms. For the RNA sequencing, we used pooled samples taken from three representative rats in each AB group. A Benjamini–Hochberg false discovery rate  assay where a higher level of latent TGF-β in the ECM was wanted. For siRNA experiments, primary foetal human cardiac fibroblasts were treated with non-targeting control siRNA or ADAMTS4 siRNA  at a concentration of 10 nM according to manufacturer’s instructions and cultured for 7 days. Conditioned cell culture medium and cellular lysates were collected for protein detection by immunoblotting and immunohistochemistry, or for quantification of TGF-β by use of tMLC, as described previously.25 Adult human cardiac fibroblasts were also exposed to mechanical stress of 10% biaxial strain at a frequency of 1 Hz for 24 h.Primary foetal and adult human cardiac fibroblasts were cultured for 7 days, as has been previously described,2.726 and reported in Supplementary Methods. The use of the samples was approved by the regional ethics committee (REK 2014/569 and 07482a).Biobanked human myocardial samples were used to determine levels of ADAMTS4 expression and activity. Samples were taken from the free wall of the left ventricle of explanted hearts from patients with dilated non-ischaemic cardiomyopathy and from donor hearts that had not been used for transplantation due to no Scandinavian recipients being available. Tissue lysates were first solubilized using the compartment protein extraction kit  according to the manufacturer’s instructions. ECM fractions were extracted as previously described2.8P < 0.05. For cell culture experiments, comparisons were performed via ANOVA for correlated samples, with Bonferroni corrections as described above. For analyses in cellular lysates and immunohistochemistry, one-way ANOVA with planned comparisons and Bonferroni post hoc corrections were performed, whereas Student’s t-tests were applied in human samples for comparisons between two groups. Log-transformation was performed to achieve equal variances (<3 standard deviation (SD) difference between groups) and/or normal distribution, as appropriate. Data are presented as mean ± 1 SD. Survival of AB rats was assessed by Cox regression and log-rank test. All statistical analyses were performed in SPSS 27 , graphs were prepared in GraphPad Prism 9 , and figure panels were assembled and illustrations prepared in Adobe Illustrator 16.0 .For comparisons between groups in the rat study, we employed one-way analysis of variance (ANOVA) with planned comparisons to detect differences between the following groups: sham vehicle vs. sham ADAMTS inhibitor, sham vehicle vs. AB vehicle, and AB vehicle vs. AB ADAMTS inhibitor. Bonferroni correction for these three comparisons was performed, and results were regarded as significant for 3.3.1Figure 1A). Following AB, echocardiographic examination of vehicle-treated rats revealed a 44% increase in left atrial diameter and a 54% increase in the E/e′ ratio. Both parameters were reduced by close to 30% in rats with AB that received ADAMTS inhibitor. This finding indicated that diastolic function and filling pressures were improved in rats treated with ADAMTS inhibitor . AB rats that were treated with ADAMTS inhibitor also demonstrated better systolic function than those treated with vehicle . Signs of advanced heart failure, i.e. the weights of the right ventricles and lungs, were not significantly different in sham compared with AB, or in AB with or without ADAMTS inhibition . Fewer rats died in the AB group treated with ADAMTS inhibitor  than in the vehicle-treated AB group , constituting a difference in total mortality  . We did not observe any clear adverse effects of ADAMTS inhibition (Supplementary Results). Overall, cardiac function was better among the surviving AB rats that were treated with ADAMTS inhibitor than among those that received vehicle.The effect of the ADAMTS inhibitor on cardiac function and remodelling was assessed in rat hearts exposed to pressure overload. The rats were placed in four groups: (i) sham rats that received vehicle (no ADAMTS inhibitor), (ii) sham rats that received ADAMTS inhibitor, (iii) AB rats that received vehicle, and (iv) AB rats that received ADAMTS inhibitor . The diameter of the left ventricle as measured by echocardiography was lower in the AB rats treated with ADAMTS inhibitor than in AB rats that were treated with vehicle . AB induced a 2.2-fold increase in collagen content compared with the vehicle-treated sham rats, and both perivascular and interstitial fibrosis were observed in AB rats . This collagen amount was reduced in the AB rats treated with ADAMTS inhibitor when compared to AB rats treated with vehicle . Overall, AB rats treated with ADAMTS inhibitor demonstrated reduced left ventricular mass and lower collagen content than AB rats receiving vehicle treatment.To determine whether ADAMTS inhibition affected cardiac remodelling, we assessed cardiac dimensions and myocardial collagen content in the pressure-overloaded rat hearts. In vehicle-treated AB rats, we observed a 64% increase in left ventricular mass and a 34% thicker left ventricular wall. AB rats that were treated with ADAMTS inhibitor exhibited a 15% lower left ventricular weight and a 13% thinner interventricular septum than the AB rats that received vehicle , while the central pro-fibrotic factor TGF-β1 was included in the list of inhibited upstream regulators . Since TGF-β is a master regulator of fibrosis,27 and 33 downstream target genes of TGF-β1 were altered by ADAMTS4 inhibition , we hypothesized that ADAMTS4 inhibition could reduce the myocardial collagen content through modulation of TGF-β. To verify this finding, we performed real-time quantitative polymerase chain reaction (RT–qPCR) on TGF-β downstream target genes in all four groups. This analysis confirmed that AB rats that had been treated with ADAMTS inhibitor exhibited significant down-regulation of genes that encoded for connective tissue growth factor, biglycan, and collagen 8a1 , all of which are genes implicated in the fibrotic response at similar timepoints post-AB.28 In contrast, we found no alterations in the expression of selected genes that encode for mediators or markers of hypertrophy or inflammation, which indicated that modulation of these disease processes was not central features of the response to ADAMTS inhibition . Appreciating the central role of TGF-β in fibrosis and heart failure development after pressure overload,27 our results suggested that ADAMTS inhibition prevented fibrosis and cardiac dysfunction by reducing TGF-β-activity.To identify disease mechanisms targeted by ADAMTS4 inhibition, we performed RNA sequencing of myocardial tissues from both groups of AB rats. In AB rats that had been treated with ADAMTS inhibitor compared with those that had been treated with vehicle, we found 120 DEGs of which 86 genes were down-regulated and 34 were up-regulated in those receiving ADAMTS inhibitor .29 To understand and characterize the role of ADAMTS4 in TGF-β signalling, we hypothesized that the cleavage of ECM components by ADAMTS4 could mobilize TGF-β. Thus, we started by determining whether ADAMTS4 could release ECM-anchored latent TGF-β. To adult human cardiac fibroblasts that produced a mature ECM over 7 days,24 we added active, recombinant ADAMTS4 with or without inhibitor for 24 h in serum-free medium before analysis. Total TGF-β levels, i.e. of both active and latent forms of TGF-β, were measured in the heat-activated, conditioned medium through the use of tMLC assay. We demonstrated that the treatment with ADAMTS4 increased the level of total TGF-β in the medium by ∼50% , whereas concomitant treatment with ADAMTS inhibitor reduced this release of TGF-β into the medium. Moreover, siRNA-mediated ablation of ADAMTS4  reduced the levels of total TGF-β in the medium in cultures of human cardiac fibroblasts . These observations suggest that latent TGF-β is transferred from the ECM to the medium upon treatment with ADAMTS4.TGF-β is stored in the ECM as a large latency complex (LLC) that is composed of LTBP1, the latency associated peptide (LAP) and the mature TGF-β-dimer . To determine whether the losses of ECM-localized fibronectin and LTBP1 were associated with proteolysis, we assessed the effects of ADAMTS4 on full-length proteins and potential cleavage fragments by immunoblotting. In ECM lysates from ADAMTS4-treated foetal human cardiac fibroblasts, we observed a 70% reduction in the amount of full-length EDA-fibronectin  and a 39% reduction in the amount of full-length LTBP1 . Also, ADAMTS4 knockdown reduced the amount of LTBP1 in the ECM . Meanwhile, fragments of these components appeared in the medium upon treatment with ADAMTS4. An 180 kDa fibronectin fragment of fibronectin and a 100 kDa fragment of LTBP1 were observed in the conditioned media after ADAMTS4 treatment, while not being clearly present in controls . Concomitant treatment with ADAMTS inhibitor counteracted the effects of ADAMTS4, reducing the loss of fibronectin and LTBP1 from the ECM and the release of fragments to the media . These observations indicate that ADAMTS4 disrupts EDA-fibronectin and LTBP1 in the ECM through enzymatic cleavage.We next sought to understand the mechanism of ADAMTS4-mediated release of ECM-bound latent TGF-β. Interactions between LTBP1 and ECM components promote TGF-β latency,3.6Figure 4A). In ADAMTS4-treated lysates, we observed a reduction in the amount of full-length fibronectin, as detected by all three antibodies, and this reduction was blunted by the addition of the ADAMTS inhibitor. Three cleavage fragments of fibronectin were observed after treatment with ADAMTS4, which were two C-terminal fragments of 30 and 70 kDa, and an 180 kDa fragment recognized by the polyclonal antibody . No fragments were detected through the use of the EDA-specific antibody. The size of the fragments indicated cleavage within or close to the EDA. This supposition was supported by additional observations in a smaller fibronectin peptide . For LTBP1, we observed a loss of full-length protein and appearance of a 100 kDa fragment in ADAMTS4-treated lysates, while only the loss of full-length protein was prevented by the inhibitor . Taken together, these results indicate that ADAMTS4 cleaves EDA-fibronectin and LTBP1, and that EDA-fibronectin cleavage is blunted by treatment with ADAMTS inhibitor.To confirm that the release of fibronectin and LTBP1 fragments was due to direct cleavage by ADAMTS4, we next incubated foetal human cardiac fibroblast lysates with ADAMTS4 alone or in combination with the ADAMTS inhibitor. To characterize the cleavage fragments at fibronectin immunoblots, we used antibodies that recognized the EDA and C-terminus, in addition to a polyclonal antibody that detected other parts of the protein . An addition of the ADAMTS inhibitor reduced the amount of EDA-fibronectin cleavage by all three enzymes at an inhibitor concentration equal to or greater than 100 nM . Versican cleavage was affected both by ADAMTS4 or -5, whereas the ADAMTS inhibitor prevented the versican cleavage by ADAMTS4, but did not prevent this action by ADAMTS5 .To determine whether the cleavage of EDA-fibronectin is specifically due to ADAMTS4 activity, we assessed the concentration–response relationship for ADAMTS4 and similar enzymes. Since ADAMTS1, -4, and -5 share proteolytic activities,3.8in vitro, we sought to explore whether this mechanism also occurred in vivo. Fibronectin cleavage was determined by immunoblotting of ECM-rich protein fractions from rat cardiac tissues. Similar to our in vitro findings, we observed an accumulation of the 180 kDa fragment of fibronectin in AB rats. This level was reduced when ADAMTS inhibitor was present . In addition, an increase in the mRNA expression of EDA-fibronectin following AB  supported the assumption that the EDA-fibronectin isoform was available in rat myocardium that was exposed to pressure overload. The myocardial ADAMTS activity in AB rats was confirmed by the accumulation of versican DPEAAE fragments, the production of which was reduced when ADAMTS inhibitor was present . A non-significant increase of the 100 kDa fragment of LTBP1 was observed in vehicle-treated AB compared to sham rats, with a non-significant reduction following treatment with ADAMTS inhibitor .Having demonstrated that ADAMTS4 cleaved fibronectin 3.9online, Table S6). Compared with healthy donor hearts, a 3.5-fold increase in the amount of 180 kDa fibronectin in the non-ischaemic failing hearts was observed . A 24-fold increase in the amount of versican DPEAAE fragments in non-ischaemic failing hearts was observed, whereas these fragments were present in negligible amounts in donor hearts . A non-significant increase in the 100 kDa fragment of LTBP1 was observed in non-ischaemic failing hearts . Taken together, these data indicate that ADAMTS4 activity, including fibronectin cleavage, is increased in human heart failure.To determine whether myocardial ADAMTS4 activity is increased during human heart failure, we assessed the level of active ADAMTS4 protein and its cleavage products in explanted hearts with dilated cardiomyopathy (DCM) , while only ADAMTS4 was up-regulated in human cardiac fibroblasts exposed to mechanical stress . The amount of 68 kDa ADAMTS4 protein, consistent with the mature form of the enzyme,36 was increased in AB rats and DCM patients, and ADAMTS5 protein was increased in AB rats, while ADAMTS1 levels were decreased in both AB rats and DCM patients . To summarize, the increased levels of ADAMTS4 seem to be consistent across different patient cohorts and models related to heart failure and cardiac fibrosis.Since ADAMTS1, -5, -9, -15, or -20 possess similar enzymatic activities as ADAMTS4, we examined their expression pattern in pressure-overloaded rat hearts, cardiac fibroblasts exposed to mechanical stress, and in failing human hearts. The amounts of ADAMTS1 and -4 mRNA were up-regulated 6 weeks after AB .In this study, we have demonstrated that (i) ADAMTS inhibition alleviates cardiac dysfunction and reduces myocardial collagen content in pressure-overloaded rat hearts; (ii) ADAMTS4 releases ECM-bound latent TGF-β partly by cleaving TGF-β-anchoring EDA-fibronectin; and (iii) ADAMTS4 is up-regulated and active in human and murine failing hearts. Taken together, these findings indicate that ADAMTS4 is a central mediator of cardiac fibrosis, and therefore, it may be a target for the treatment of patients with cardiac fibrosis and heart failure  or have been reported in unchallenged ADAMTS4 KO mice,66 exposing the vasculature to stress during AB may affect aortic remodelling. Therefore, future studies on the roles of ADAMTS4 in cardiac disease should include blood pressure measurements and aortic examinations.Our findings should be interpreted in light of the limitations of the study. First, although we did not observe any adverse effects of ADAMTS4 inhibition in our study, infrequent events or off-target effects cannot be excluded. However, repeated administration of a similar compound did not lead to adverse effects in guinea pigs.67 and TGF-β represents an attractive therapeutic target. However, direct inhibition of TGF-β-signalling has proved to have ambiguous effects in rats exposed to pressure overload due to detrimental effects on wound healing.68 Therefore, approaches to inhibit the detrimental actions of TGF-β without interfering with its physiological roles are warranted.69 The findings that ADAMTS4 is up-regulated in heart failure and that it cleaves EDA-fibronectin, which is an ECM component mainly seen in diseased tissue, strengthen the idea that ADAMTS4 should be a specific target in TGF-β modulation and anti-fibrotic treatment in heart disease. Although fibrosis occurs in most forms of heart failure,67 the activity of ADAMTS4 in other phenotypes than those examined in our study should be clarified to further support the translational potential of these findings.Fibrosis is a central characteristic of heart failure of different phenotypes and aetiologies,Cardiovascular Research online.Conceptualization: M.V., P.M.E., A.S., A.R., C.R.C., J.M.A., I.S., G.C. Methodology: M.V., P.M.E., A.S., E.S.N., K.B., A.R., A.O.M., J.M.A., I.S., G.C. Validation: M.V., P.M.E., A.S., A.R., I.G.L., L.Z., M.B.Ø., J.Ø. Formal analysis: M.V., P.M.E., I.G.L. Investigation: M.V., P.M.E., A.S., E.S.N., K.B., A.R., I.G.L., L.Z., M.B.O., J.Ø., C.R.C., C.H.W., J.R., C.P.D., A.E.F., I.M.H.I., E.E., A.O.M., T.T., J.M.A., I.S., G.C. Resources: M.V., E.S.N., K.B., L.Z., C.H.W., J.R., C.P.D., A.E.F., I.M.H.I., E.E., T.T., J.M.A., I.S. Writing—original draft: M.V., G.C., P.M.E. Writing—review & editing: all. Visualization: M.V.Supervision: M.V., G.C. Project management: M.V., G.C. Funding acquisition: M.V., G.C.cvad078_Supplementary_DataClick here for additional data file.


The choice of a vascular graft in patients with femoral atherosclerosis has always been a matter of debate. But when a deep review of the literature is done, the autogenous graft using the saphenous vein is considered the most reliable graft material for the vessels involved below the level of the inguinal ligament. In recent years, there have been published studies comparing vascular versus prosthetic grafts. We report a case covering a similar domain where femoropopliteal bypass was done using a polytetrafluoroethylene (PTFE) prosthetic graft and the outcome after the surgical procedure. Bypass procedures are considered a procedure for salvaging a limb in patients suffering from atherosclerotic changes in vessels that can produce peripheral arterial ischemia . It is vThe choice of graft, whether venous or prosthetic, depends on several factors, which vary from patient to patient and from surgeon to surgeon. Patient factors include the unavailability of an adequate saphenous venous graft and financial conditions, while a surgeon's factors include preference and expertise in dealing with such grafts . We repoA 53-year-old non-diabetic man, smoker, and hypertensive presented to the cardiothoracic and vascular surgery outpatient department with the chief complaints of intermittent claudication in his right leg for six months. On examination, peripheral pulses like popliteal, posterior tibial, and dorsalis pedis were non-palpable in the right leg, and the limb was cold with overlying skin changes and capillary refill time of more than three seconds, and brittle nails in comparison to the contralateral limb. In the left lower limb, the peripheral pulses were palpable, capillary refill time was less than three seconds, and there were no overlying skin changes with the normal texture of nails.The patient's pulse was 80 beats per minute, regular in rhythm, and the blood pressure was 120/70 mm Hg on antihypertensive medications. His pulses were palpable in both upper limbs. Motor and sensory functions were intact in both lower limbs. A duplex study was obtained in assessing the bilateral lower limbs, which revealed 100% occlusion of the femoropopliteal segment in the right lower limb and normal venous systems in both lower limbs. The ECG and 2D echo were also done and the findings were within normal limits.The computed tomography angiography findings in the left lower limb were within normal limits, while in the right lower limb, there revealed a complete filling defect of the superficial femoral artery at the level of the mid-thigh with distal recanalization just above the level of the knee, features suggestive of thrombosis. The right popliteal, anterior tibial and posterior tibial arteries show normal contrast opacification, as shown in Figures After subjecting the patient to all these investigations, a femoropopliteal bypass for the right lower extremity was recommended to the patient using a PTFE graft. The patient was shifted to the operation theater and placed under epidural anesthesia in a supine position. Initially, urethral catheterization was done. Painting and draping were done from the umbilicus down to the mid-calf region. A skin incision was made in the right mid-thigh area along the course of the superficial femoral artery, and arterial vessels were isolated. An incision was extended along the distal femoral artery up to the adductor canal to expose the popliteal artery above the knee.Systemic heparinization with a bolus of 5,000 units of unfractionated heparin was achieved. The proximal and distal controls were established. The proximal right superficial femoral artery and right distal popliteal artery bypass graft was placed using 6-0 polypropylene sutures and a 6-mm PTFE graft as venous graft was insufficient in this patient due to varying anatomy of the venous vasculature, thereby establishing a femoropopliteal bypass graft from the right distal superficial femoral artery to the right popliteal artery, just above the knee.It was ensured that there would be no twisting or kinking of the graft. An intravenous sodium bicarbonate injection was given just before releasing the arterial control clamps. Good flow in the graft was ensured before closing the wound. The wound was closed with a drain kept in situ, which was removed on postoperative day 2. All peripheral pulses demonstrated strong pulsation, with oxygen saturation of 100% in both lower limbs. The patient was kept on intravenous heparin with dextran infusion  for three days.Thereafter, the patient was switched to oral antiplatelet, statins and anticoagulant therapy. His postoperative recovery was uneventful. The claudication improved and disappeared 10 days postoperatively. The patient was discharged on the 14th postoperative day with the advice of regular dressing changes, continue the prescribed medications, and to be seen at a follow-up visit. The placement of the prosthetic graft is shown in Figure In this case, we have convincingly learned that in patients where the vascular grafts, such as great saphenous venous grafts, are not sufficient to carry out bypass procedures because of the narrow veins and varying vasculature, prosthetic grafts like PTFE may be employed to carry out limb salvage bypass surgeries in order to restore vascularization .The expected postoperative complications can include graft thrombosis, bleeding, and local site wound infection. The most important marker regarding the success of such bypass surgery is the ultimate patency of the graft as demonstrated with adequate vascular flow through the graft .And as far as patency is concerned, such grafts used in femoropopliteal bypass surgeries have shown the best outcomes, with around 90% of these grafts remaining patent for years, but the same cannot be taken as the principle for symptomatic control as most of the patients might complain of intermittent symptoms postoperatively . FemoropWe are reporting this case to elicit the successful management of a patient presenting with occlusive vascular ischemic disease and how timely intervention and the use of a prosthetic PTFE graft was utilized because the greater saphenous vein as a graft was not acceptable in this patient. This case may sensitize the surgeons performing the vascular bypass surgery using conventional methods to the fact that, apart from the vascular grafts, these prosthetic grafts also have outstanding outcomes when revascularizing a lower limb and demonstrate rapid improvement post-operatively, as observed in our patient.


We present two cases of transmission of a pancreatic adenocarcinoma from a single donor to two kidney transplant recipients. Autopsy of the donor revealed a pancreatic adenocarcinoma that had already spread locally to the regional lymph nodes and had not been detected at the time of organ procurement. Both recipients were carefully monitored, as neither consented to graft nephrectomy. In one patient, the tumor was discovered on surveillance biopsy of the graft approximately 14 months after transplantation, and in the second patient, ultrasound-guided aspiration needle biopsy of a growing formation in the lower pole of the graft revealed poorly differentiated metastatic adenocarcinoma. Both patients were successfully treated with graft nephrectomy and complete discontinuation of immunosuppression. None of the follow-up imaging showed persistent or recurrent malignancy, and both patients were candidates for re-transplantation. These exceptional cases of donor-derived pancreatic adenocarcinoma suggest that removal of the donor organ and restoration of immunity may lead to complete recovery. Here, we report one of the rarest cases of transmission of adenocarcinoma of the pancreas from a deceased single multiorgan donor to two kidney transplant recipients and its treatment after both patients refused removal of the kidney graft.2.2.1.The donor was a 46-year-old woman who died of spontaneous intracerebral hemorrhage. She had no known history of malignancy nor was there any evidence of malignancy listed in the Eurotransplant donor report. The other organs transplanted were the liver, heart, and lungs. Due to the patient’s history of diabetes, simultaneous transplantation of the kidney and pancreas was not considered.2.2.The recipient of the right kidney was a 61-year-old man and the recipient of the left kidney was a 40-year-old man. Both kidney transplant recipients had been treated with dialysis for approximately 3 years  and were successfully transplanted with a standard immunosuppression protocol  in late November 2015. Methylprednisolone was discontinued in both patients after 1 week, and they were discharged with improving graft function (serum creatinine (SCr) of 161 μmol/l in the right kidney transplant recipient and 169 μmol/l in the left kidney transplant recipient).On December 21, less than 1 month after transplantation, our center was informed by Eurotransplant that the donor had adenocarcinoma of the pancreas, which had already spread locally to the regional lymph nodes. The tumor was not macroscopically detectable at the time of donation and was only discovered at autopsy and later confirmed histologically. Because hematogenous spread of the pancreatic tumor appeared possible premortem, both recipients were offered immediate explantation of the graft. Unfortunately, neither patient consented to this procedure because of the tremendous burden of dialysis treatment. At this point, both patients were switched from mycophenolate to everolimus and dual immunosuppression with low-dose tacrolimus was continued. Both patients were followed-up closely. The timing of most important events for both recipients is presented in During the following year, the recipient of the right kidney was monitored with regular ultrasound examinations of the transplanted kidney and with the tumor marker carbohydrate antigen (CA) 19–9, and all test results were within the reference range. He declined monitoring with computed tomography (CT) because of the possibility of contrast-induced acute kidney injury. 12 months after transplantation, he was switched back to mycophenolate because of worsening lower extremity edema. A surveillance biopsy 14 months after transplantation revealed poorly differentiated adenocarcinoma in two of four tissue samples. Immunohistochemical findings  suggested a pancreatic origin of the tumor . A samplIn the left kidney recipient, baseline contrast CT scans of the thorax and abdomen revealed no evidence of tumor. CA 19–9 was within the reference range. Follow-up abdominal ultrasound and CA 19–9 testing in the first year after transplantation were within the reference range. Approximately 12 months after transplantation, a graft biopsy was performed per protocol and indication (due to increasing proteinuria of more than 2 grams per day), which showed evidence of everolimus toxicity, leading to discontinuation of everolimus and reintroduction of mycophenolate. In February 2017, 14 months after transplantation, follow-up imaging diagnostics were performed, including CT of the abdomen and thorax, an MRI of the head, and an FDG-PET. The latter showed increased uptake of radiotracer in the anterior abdominal wall, at the site where a peritoneal dialysis catheter had previously been inserted; an aspiration needle biopsy was negative for tumor cells. The remaining examination findings were unremarkable. In summer and fall 2017, serial ultrasound examinations of the graft showed growing complex formation in the lower pole of the kidney . In OctoExamination of the explanted kidney revealed an adenocarcinoma with identical immunohistochemical features as in the first recipient, suggesting a pancreatic origin of the tumor . Further2.3.At the time of discovery of pancreatic adenocarcinoma, all other organ recipients were in good health. However, except for the heart transplant recipient, their long-term survival was poor. The liver transplant recipient was immediately retransplanted but later died of metastatic pancreatic adenocarcinoma. The lung transplant recipient was also retransplanted and died 18 months after retransplantation from chronic rejection and multiorgan failure. Data on the other organ recipients were provided by Eurotransplant because they were treated at different European institutions.3.The cases we present are among the rare reports of pancreatic tumor transmission in organ transplantation that were successfully treated without recurrence of the tumor in any kidney recipients. Our patients were two adult men who refused removal of the kidney graft after autopsy of the donor revealed adenocarcinoma of the pancreas. Both were monitored closely, which allowed early detection of tumor transmission. Subsequent removal of the kidney graft and discontinuation of immunosuppression led to complete recovery in both recipients.Transplantation of organs from donors with pancreatic cancer is considered an unacceptable risk because of its aggressive behavior and high risk of spread to other organs. Our donor was a 46-year-old woman with two known risk factors for pancreatic cancer, diabetes mellitus and cigarette smoking . Becausede novo malignancy in kidney graft recipients even in patients receiving combination therapy with cyclosporine/tacrolimus  as a possible donor-related malignancy in three recipients . PancreaIn conclusion, our two cases show that even the unfortunate event of transmission of adenocarcinoma of the pancreas in kidney transplant recipients can be successfully treated without recurrence of the tumor. In a kidney graft lesion where tumor transmission is suspected, an ultrasound-guided biopsy is recommended for pathohistological evaluation. If preventive graft nephrectomy is not an option, immunosuppressive therapy should be adjusted and patients should be monitored radiologically on a regular basis, at least by periodic ultrasonography of the graft. Patients who have had a tumor transferred with the kidney graft in the past can be successfully placed on the waiting list for retransplantation.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.TBM: writing. GM, MO, AK, and MA: conceptualization, supervision, and writing. JB-P: supervision. NK and MF: pathology report, writing, and pathology supervision. SH: surgical supervision. TM: oncology supervision. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The addition of toxic flame retardants to commercially available polymers is often required for safety reasons due to the high flammability of these materials. In this work, the preparation and incorporation of efficient biodegradable starch-based flame retardants into a low-density polyethylene (LDPE) matrix was investigated. Thermoplastic starch was first obtained by plasticizing starch with glycerol/water or glycerol/water/choline phytate to obtain TPS-G and TPS-G-CPA, respectively. Various LDPE/TPS blends were prepared by means of melt blending using polyethylene graft maleic anhydride as a compatibilizer and by varying the content of TPS and a halogenated commercial flame retardant. By replacing 38% and 76% of the harmful commercial flame retardant with safe TPS-G-CPA and TPS-G, respectively, blends with promising fire behavior were obtained, while the limiting oxygen index (LOI ≈ 28%) remained the same. The presence of choline phytate improved both the charring ability and fire retardancy of starch and resulted in a 43% reduction in fire growth index compared to the blend with commercial flame retardant only, as confirmed by means of cone calorimetry. Standard UL 94 vertical tests showed that blends containing TPS exhibited dripping behavior (rated V2), while those with commercial flame retardant were rated V0. Overall, this work demonstrates the potential of starch as a natural flame retardant that could reduce the cost and increase the safety of polymer-based materials. Despite their good flame-retardant performance, halogen containing FRs are not good candidates from an environmental point of view due to their high toxicity, and some of them were banned a few decades ago [Polyethylene (PE) is one of the most widely used polyolefins in the global plastics market around 34%)  and is a4%  and iades ago . In viewades ago ,13. Seveades ago , β-cycloades ago , organicades ago , phytic ades ago , polydopades ago , have beg-GMA) or LDPE-grafted maleic anhydride (LDPE-g-MA) [Using starch as an FR is a wise and promising option because this natural polymer is safe, abundant, and inexpensive. Starch from various sources has been successfully blended with LDPE via extrusion, resulting in blends with inferior mechanical properties in comparison with neat LDPE . In factPE-g-MA) . UnfortuThe aim of this work was to develop a new starch-based flame retardant that can be used in PE formulations. In this way, it will be possible to obtain a low-cost and partially biodegradable product and reduce or eliminate the need for harmful flame retardants. For this purpose, various LDPE/TPS blends with different proportions and compositions of TPS were prepared by means of melt blending. The effect of combining the biobased FRs developed in this work with a commercial halogenated flame retardant in LDPE blends was also investigated. The materials were analyzed using scanning electron microscopy (SEM), tensile tests, thermogravimetric analysis (TGA), the limiting oxygen index (LOI), flammability (UL 94) and cone calorimetry.3, Eurisotop, Saarbrücken, Germany), deuterated dimethyl sulfoxide , deuterated water , glycerol , phytic acid  and sodium hydroxide  were used as received.Choline chloride , ethanol absolute , deuterated chloroform (CDClIsoAdditive FR L069 flame retardant (brominated-based additives in LDPE carrier) was kindly supplied by Isolago  and used as received.TM) and polyethylene-graft-maleic anhydride  were kindly supplied by Componit  and used as received.Potato starch, low-density polyethylene (LDPE) with a melting temperature of 140 °C .TPS and LDPE/TPS blends were prepared by means of melt blending using a laboratory mixer HAAKE1H nuclear magnetic resonance (1H NMR) spectra of the compounds using a Bruker Avance II 400 MHz Spectrometer  with a 5 mm TIX triple resonance detection probe.We recorded 400 MHz −1, with 4 cm−1 spectral resolution and 64 accumulations, at room temperature using an Agilent Technologies Carey 630 spectrometer  equipped with a golden gate single reflection diamond ATR.Fourier transform infrared (FTIR) spectra were obtained in the range of 4000 to 750 cmThe mechanical properties of the blends were accessed via tensile tests using a Chatillon TCD100 machine  at 5 mm/min speed and a load cell of 5 kN, with at least 5 specimens (10 ± 0.5 mm × 70 ± 0.5 mm).Samples for scanning electron microscopy (SEM) analysis were prepared using the fraction surface of the tensile test’s specimens. The fracture surface was coated with gold and analyzed in a Field Emission SEM ZEISS MERLIN Compact/VP Compact Gemini II .Vertical flammability assays were conducted by AIMPLAS (Spain) following the UL 94:2013 standard .LOI assays were conducted by AIMPLAS (Spain) using the Netzsch-Taurus Oxygen  system following the standard UNE-EN ISO 4589-2:2017 . Average2): 50; orientation: horizontal; separation (mm): 25; wire grid: No; N of specimens: 3; average sample thickness (mm): 1.5; pre-conditioning 48 h at 23 ± 2 °C, 50 ± 10%RH; environmental conditions: 23 ± 2 °C; 50 ± 10%RHCone calorimeter tests were conducted by AIMPLAS (Spain) using the FTT ICONE CLASSIC machine  and following the standard EN ISO 5660-1:2015 . Conditi−1, under nitrogen purge flow (sample weight in the range of 5 to 10 mg).Thermogravimetric (TG) analyses were carried out using a NETZSCH STA 44F5 , from 20 °C to 500 °C, at a heating rate of 10 °C·min1H NMR spectroscopy  and TPS-G-CPA . All compounds were placed in a plastic flask and mixed manually with a spatula. The flask was sealed and the mixture was allowed to stand overnight. The mixture was then processed via melt mixing at 120 °C (TPS-G) or 140 °C (TPS-G-CPA) for 5 min, at 100 rpm.g-MA compatibilizer, and a commercial flame retardant  were added to a mixing chamber. The mixtures were processed at 140 °C with a rotation speed of 100 rpm for 5 min. The obtained mixtures were cut into small pieces by hand, placed between two silicone squares and pressed in a Carver® hydraulic press under 0.5 bar at 140 °C for 5 min, followed by cooling at room temperature. The compounds were produced in the form of sheets with a thickness of approx. 5 mm.To prepare LDPE/TPS blends, LDPE, TPS, PE-g-MA compatibilizer was also incorporated into the formulation during the melt blending process  were prepared by means of melt blending at 120 °C and 140 °C, respectively, when TPS-G or TPS-G-CPA was used as the biobased flame retardant  into the LDPE/TPS-G(-CPA) blends prepared in this work was investigated. −1, 2851 cm−1, 1468 cm−1 and 718 cm−1, corresponding to CH2 asymmetrical stretching, CH2 symmetrical stretching, bending deformation and rocking deformation, respectively [C−O stretching at 920 cm−1, 1022 cm−1 and 1148 cm−1, bound water at 1648 cm−1, hydroxyl groups at 3277 cm−1, CH stretching at 2914 cm−1 and glycerol at 1423 cm−1 [g-MA and TPS-G was confirmed by the disappearance of the symmetric and asymmetric maleic anhydride groups of PE-g-MA at 1714 cm−1 and 1791 cm−1, respectively, in the FTIR spectrum of the blend /FR69 blends as flame retardant materials. The tests were conducted in the same manner as previously described for LDPE/TSP-G(-CPA) blends, but this time the materials were re-ignited with a flame after the initial quenching, and the time until the flame was extinguished was recorded as the second quenching time. Compared to LDPE/TPS-G(-CPA) blends , LDPE-baBlends with higher TPS content and higher flame retardancy in preliminary burning tests (47.5 LDPE/38 TPS-G/9.5 FR69 and 47.5 LDPE/19 TPS-G-CPA/28.5 FR69) were selected for further characterization and evaluation of their flame retardancy. LDPE with 50 wt% commercial flame retardant (50 LDPE/50 FR 69) was used as a reference material. The LOI can provide information about the relative flammability of polymers, as it indicates the minimum percentage (vol%) of oxygen in the atmosphere that can sustain a flame on a material. Therefore, the higher the flammability of the material, the lower the LOI value. It is noteworthy that the samples in which a portion of the commercial flame retardant was replaced with the starch-based products developed in this work have a similar LOI value to the reference material that contained only the commercial flame retardant . All tes2). The values obtained for time to ignition (TTI) values in The fire behavior of LDPE/flame retardant blends was evaluated using cone calorimetry, which mimics realistic fire conditions and provides several valuable parameters, particularly the rate of heat release rate during combustion. To increase the flame resistance of LDPE/flame retardant blends and reduce flame spread, it is desirable to have a low total heat release (THR) or a low peak of heat release rate (pHRR) . The resT ≈ 128 °C), (ii) degradation of glycerol (T ≈ 270 °C), (iii) degradation of starch, CPA, and FR69 (T ≈ 350 °C), and (iv) degradation of LDPE (T ≈ 475 °C) [T5wt% and T10wt% of the samples  ,37. The  samples . In addi samples . These rg-MA as a compatibilizer. The results of FTIR spectroscopy and SEM analysis showed that there was good compatibilization between starch and LDPE. However, this was less effective when TPS-G-CPA was used. Nevertheless, the mechanical properties of both blends were similar in terms of tensile strength. The plasticizing effect of CPA was confirmed by an increase in elongation at break of the LDPE/TPS-G-CPA blends compared to those with TPS-G. Acceptable fire behavior was achieved by combining the starch-based FR with a commercial flame retardant (FR69), with the most promising blends being 47.5 LDPE/38 TPS-G/9.5 FR69 and 47.5 LDPE/19 TPS-G-CPA/28.5 FR69. These blends had a similar LOI (about 28%) to that of reference blend containing only the commercial flame retardant (50 LDPE/50 FR69). CPA not only imparts higher plasticity to the starch, but also improves the charring ability, flame retardancy, and effective combustion component content of the blends, as well as a 43% reduction in fire growth index compared to the reference blends, suggesting that it may contribute to lower fire spread in the event of a fire. These results indicate that starch plasticized in the presence of water, glycerol and CPA has a promising future as a safe and cost-effective flame retardant for LDPE formulations.In this work, the possibility of using starch as a natural flame retardant for LDPE was investigated. Different blends of LDPE/TPS g and LDPE/TPS-G-CPA were prepared by means of melt blending in the presence of PE-


The in vitro digestibility of potato starch-based foods interacting with milk protein and soybean oil was investigated. Microstructures and rheological changes upon digestion were determined. The results showed that the addition of milk proteins (casein and whey protein) promoted gelatinized potato starch digestion, while soybean oil slowed down gelatinized potato starch digestion. A mixture of soybean oil and milk protein promoted the digestion of milk protein, while a mixture of gelatinized potato starch and milk protein inhibited the digestion of milk protein. The mixture of milk protein and/or gelatinized potato starch with soybean oil promoted the release of free fatty acids in soybean oil. The highest release rate of free fatty acids was attained by a mix of milk protein and soybean oil. The mixed samples were digested and observed with a confocal laser scanning microscope. The viscosity of the digestates was determined by a rheometer. Overall, the results demonstrated that the addition of milk protein and soybean oil had an effect on the in vitro digestibility of gelatinized potato starch and its microstructure. Starch is a major food source for humans, accounting for about 70 percent of the calories in the human diet. Starch-based foods are products containing high amounts of starch, but these foods often also contain a certain number of proteins and fats. There are many studies on the binary interaction between starch and proteins or lipids ,2,3,4. MThe influence of proteins and lipids on the starch digestibility of kodo millet flour and rice flour was investigated . The stuStarch-based foods such as bread, mashed potatoes, and noodles contain not only starch but also protein and lipids. Processing conditions and the composition of raw materials could affect the microstructure, rheological characteristics, and digestibility of these foods. Food processing could influence the glycemic index (GI) of potato starch products , while tThus, this study focused on the effect, in terms of interactions, of adding CA or WP and SBO to gelatinized potato starch (GPS) on GPS properties: changes in GPS microstructure, GPS rheology, and GPS digestibility. In addition, the effect of CA, WP, and SBO on starch hydrolysis in GPS was studied, along with its mechanism. New findings result from this study, which could benefit food manufacturing of potato starch in terms of applying such results to design a more suitable process and product in the future.w/w) total protein was purchased from Vicchi Enterprise Co., Ltd. . WP with 80% total protein was received from Shanghai Yuanye Biological Technology Co., Ltd. . SBO  used in this study was bought from a convenience store in Nakhon Ratchasima province, Thailand. α-Amylase from porcine pancreas , pepsin from porcine gastric mucosa (P7000), and pancreatin from porcine pancreas  were bought from Sigma-Aldrich Chemical Co. . Megazyme International Ireland Ltd.  provided the assay kit of glucose oxidase-peroxidase (GOPOD), the assay kit of total starch, and amyloglucosidase (3200 U/mL). All chemicals used in this study were of analytical grade. Potato starch (PS) was provided by Bangkok Inter Food Co., Ltd. . CA powder with 82% . Fatty acids (FAs) of the SBO were determined according to the method of AOAC 969.33 (2000) by Gas chromatography . The initial amount of starch in the samples was evaluated using an analytical kit for total starch. The potato starch-based foods were prepared according to the previous research methodology of Guan et al. . BrieflyA rapid in vitro starch digestion assay following the methods of Sopade and Gidley was used . Test saHS was the percentage of starch hydrolysis , hS was the amount of starch hydrolyzed, iS was the initial amount of starch (g), and Gp was the amount of glucose produced (g). Generally, 0.9 was used as the conversion factor from starch to glucose, which was calculated from the molecular weight of starch monomer/molecular weight of glucose (162/180 = 0.9). Aliquots (0.5 mL) were withdrawn at 0, 10, 20, 30, 45, 60, 90, 120, 150, 180, 210, and 240 min of digestion during the incubation in the intestinal stage, followed by mixing with 95% ethanol (3 mL). The aliquots were then analyzed for glucose using the GOPOD-assay kit. The starch hydrolysis was calculated with the following equation [tD (g/100 g dry starch) was the digested starch at time 0t, D was the digested starch at time t = 0, D∞ was the digestion at infinite time (0D + ∞−0D), and K was the rate constant (min−1). ∞−0D was estimated from t = 0–240 min.The digestogram showing digested samples at a specific time period was modeled using Equation (2) .(2)Dt=DGIs of the samples, the areas under the digestograms (AUCexp) were computed with Equation (3) [In order to calculate the estimated tion (3) :(3)AUCexHI) of each test sample was determined by dividing the area under the digestogram of the sample with the area under the digestogram of fresh white bread, which was around 17,000 min g/100 g dry test sample (from 0 to 240 min) in this study. Using the parameters of the modified first-order kinetic model for the test samples and fresh white bread, GI (average) was estimated (GIAVG) for each sample and was calculated using Equation (4) [Estimated GI values were determined following the method of Goñi with some modifications . The esttion (4) :(4)EstimThe in vitro protein digestibility of the test samples was carried out following the method of Srigiripura and Kotebagilu et al. . All tesThe intestinal digestion model was modified slightly from the methodologies of Hu et al., Qin et al., and Wan et al. ,29,30. TNaOH for sampleV was the volume of NaOH (L) titrated into the reaction vessel to neutralize the FFAs released; NaOH for blankV was the volume of NaOH (L) titrated into the reaction vessel to neutralize the FFAs released in the absence of oil. NaOHC was the concentration of NaOH (0.1 M); lipidW was the initial mass of SBO (g) in the intestinal phase; and lipidMW was the average molecular weight of corn oil (872 g mol−1). Here, Samples prepared in 2.3 were taken for microstructure analysis during the simulated digestion process, in which these samples were digested in the in vitro stomach stage for 15 min and in the in vitro small intestine stage for 5 min. The changes in these samples were observed with confocal laser scanning microscopy (CLSM) using the fluorescent mode  according to the method of Thaiudom and Pracham . APTS . An analysis of variance (ANOVA) was carried out to determine differences. A w/w. Protein was the main component of CA and WP, and the protein content was 81.25 and 78.67% w/w, respectively. The main component of SBO was 99.9% w/w fat. The effect of MP addition (CA or WP) and/or SBO on the starch digestibility of GPS is shown in When pancreatin and amyloglucosidase were added to the test sample, GPS/CA, GSP/WP, GSP/CA/SBO, and GSP/WP/SBO were rapidly digested within the first 30 min. At 30 min, starch digestibility reached 30–40%. The digestion rate of GPS and GPS/SBO during this period was relatively slow, and the digestibility was less than 10% after half an hour. These differences might be due to the composition of the individual test samples ,36. The kinetic constant (k), the hydrolysis index (HI), and the estimated glycemic index (eGI). The important factor in the glycemic response was the rate of starch loosening. The in vitro investigations predicted postprandial results with good accuracy. The addition of SBO had no significant effect on the kinetic constants of GPS, while the addition of MP to GPS significantly increased the kinetic constants of GPS. The viscosity of the samples that contain PS was measured. It can be seen from the results that the smaller the viscosity, the larger the kinetic constant, and the easier the GPS was digested, the higher the eGI value. When MP was added to GPS, the voided spaces could be seen in the three-dimensional structure of GPS. This assisted α-amylase to penetrate GPS more easily while SBO infiltrated the GPS 3-D structure, which might have led to hydrophobic interaction between GPS and SBO. Thus, GPS was more tightly bound to SBO, and sequentially, the α-amylase rarely digested the starch a,b,d.β-lactoglobulin and β-casein were located at the oil-water interface. They were more easily broken down by pepsin when adsorbed at the oil-water interface than in solution [CA, WP, GPS/CA, GPS/WP, CA/SBO, WP/SBO, GPS/CA/SBO, and GPS/WP/SBO possess protein contents of 0.990%, 0.987%, 0.958%, 0.959%, 0.939%, 0.997%, 0.971%, and 0.934%, respectively. The protein content of all samples was close to 1.0 % and not significantly different among those samples. The protein digestibility of the test samples is shown in solution . The prosolution . On the solution . For theThe fat digestibility of all samples containing SBO was determined. Total FFAs released from the oil phase are shown in All samples containing GPS and the state of each sample in the process of simulated intestinal conditions were observed by CLSM. The observation results are shown in In the binary system, regarding the GPS/CA and GPS/WP samples, it can be seen from The observation results of GPS/SBO are shown in Regarding the ternary system, the images of GPS/CA/SBO e and GPSThe viscosity of the test samples containing GPS during the simulation of in vitro digestibility is shown in With the addition of the enzymes prior to the simulated digestion in the intestinal stage, the viscosity of the sample in the reaction tube decreased, not only because the test sample was diluted by the addition of the enzyme solution but also because of the rapid hydrolysis of the sample by the addition of enzymes. Compared with Control-GPS, the viscosity of each sample with the added enzyme solution was significantly lower due to the hydrolysis of GPS, SBO, and MP with the addition of specific enzymes. GPS was hydrolyzed by α-amylase and amyloglucosidase to sugar, dextrins, and maltooligosaccharides, which possess a lower viscosity than GPS . SBO wasThe addition of MP and/or SBO to GPS changed the digestibility properties of the GPS. SBO inhibited the digestion of GPS, while the addition of MP promoted the digestion of GPS. The mixture of SBO and MP (CA or WP) promoted the digestion of protein, while the mixture of GPS and MP inhibited it. The mixtures of GPS/MP/SBO inhibited protein digestion. However, this inhibition was found to be at a higher rate in the sample of GPS/SBO than in GPS/MP/SBO. A mixture of MP and/or GPS with SBO promoted the release of FFA in SBO, while a mixture of MP with SBO resulted in the largest release of FFA in SBO. CLSM was an effective tool for revealing the changes in the binary and ternary systems during simulated digestion. Mixing SBO with GPS increased the viscosity of GPS, while mixing MP with GPS decreased the viscosity of GPS, resulting in inhibiting and increasing GPS digestibility, respectively. In conclusion, the evidence found in this study could be of use as guidance for food processing and food product development, especially in the field of food ingredient interaction in dairy and starch technology.


Monolateral pin-to-bar-clamp fixators are commonly used to stabilize acute extremity injuries. Certain rules regarding frame geometry have been established that affect construct stability. The influence of sagittal pin angulation on construct stiffness and strength has not been investigated. The purpose of this biomechanical study was to demonstrate the effect of a pin angulation in the monolateral fixator using a composite cylinder model. Three groups of composite cylinder models with a fracture gap were loaded with different mounting variants of monolateral pin-to-bar-clamp fixators. In the first group, the pins were set parallel to each other and perpendicular to the specimen. In the second group, both pins were set convergent each in an angle of 15° to the specimen. In the third group, the pins were set each 15° divergent. The strength of the constructions was tested using a mechanical testing machine. This was followed by a cyclic loading test to produce pin loosening. A pull-out test was then performed to evaluate the strength of each construct at the pin–bone interface. Initial stiffness analyses showed that the converging configuration was the stiffest, while the diverging configuration was the least stiff. The parallel mounting showed an intermediate stiffness. There was a significantly higher resistance to pull-out force in the diverging pin configuration compared to the converging pin configuration. There was no significant difference in the pull-out strength of the parallel pins compared to the angled pin pairs. Convergent mounting of pin pairs increases the stiffness of a monolateral fixator, whereas a divergent mounting weakens it. Regarding the strength of the pin–bone interface, the divergent pin configuration appears to provide greater resistance to pull-out force than the convergent one. The results of this pilot study should be important for the doctrine of fixator mounting as well as for fixator component design. External fixators are widely used in trauma and orthopedic surgery. In its elementary form, a fixator construct consists of bone pins, couplings, and rods placed by the surgeon on a unilateral frame used to stabilize acute severe limb injuries ,2,3. ForSuccessful outcomes in both temporary and definitive fracture treatment depend on a stable fixator construct. While axial micromotion in the fracture gap favors callus formation, excessive movement is detrimental and can lead to secondary reduction loss, pin loosening, and malunion ,6,7,8,9.Another quality of a fixator–bone construct is its strength, defined by its ability to maintain integrity against forces. Emphasis is placed on the pin–bone interface because it is the weakest link in the fixator–bone chain and, in addition to pain and loss of reduction, pin loosening appears to promote pin tract infection ,18,19. IThe main objective of this mechanical study was to investigate the influence of sagittally oriented pin angulation on construct stiffness in a monolateral fixator. Our clinical experience suggests that a configuration with converging pin pairs has higher construct stiffness and a configuration with diverging pin pairs has lower construct stiffness compared to a parallel configuration.The second objective was to evaluate the strength of the different constructs at the pin–bone interface by pull-out testing. We hypothesized that the pull-out resistance of the angled pin pairs would be higher than that of the parallel version. In addition, a pin loosening condition was to be induced prior to the pull-out test in which the angled pin pairs would show an even greater superiority.Hollow short fiber reinforced epoxy cylinders with an outer diameter of 30 mm, a wall thickness of 4.3 mm, and a length of 500 mm  were chosen as the bone analog. The goal was to create a basic model comparable to a mid-diaphyseal tibial bone. Cylinders were preferred to anatomical composite tibial bones for more general results and greater accuracy in reproducibility of the fixator–bone constructs. The cylinders were cut into 155 mm segments using a band saw, each representing a fragment of a simulated diaphyseal comminuted fracture gap model.Three types of fixators were created that differed in their pin-pair configuration. The first type had parallel pins, the second type had converging pins with an inter-pin angle of 30°, and the third type had diverging pins with an inter-pin angle of 30° in the sagittal plane. Three constructs of each type were mounted and evaluated for a total of nine specimens.The cylinder segments were loaded with self-drilling half pins  using a custom-made steel device that allowed either perpendicular or sloped pin insertion at an angle of 15 degrees to the cylinder surface . BecausePin–rod couplings and carbon connecting rods (8 mm × 350 mm) from a previously clinically used Hoffman II external fixation system  were set up for a basic monolateral, monorod fixator frame. Frame offset and fracture gap size were held constant at 50 and 20 mm, respectively, according to ASTM F1541-17 using a jig made of multiplex boards. A torque wrench was used to tighten the couplings to a torque of 15 Nm. All fixator parts components, except the deformed pins, were reused throughout the test.The bone models were suspended on the testing machine using ball joints . These suspensions were reused, since their one part was molded of cylindrically shaped plaster adjusted to the inner diameter of the composite cylinders and thus pluggable, while the further part was attached to the testing machine over intercalated multiplex boards . The difAll tests were performed on a uniaxial mechanical testing machine  and were based on ASTM F1541-17. Each specimen was subjected to the following mechanical tests: For a quasi-static axial load test, a peak force of 200 N was chosen because it simulates a partially weight-bearing condition often prescribed for patients with a tibial external fixator. The minimum load was set at 20 N. The load was applied at a rate of 40 N/s. Ten load cycles were performed, the first five being preconditioning cycles for subsequent exclusion. Load and displacement data were recorded by the machine’s transducer for later determination of the corresponding construct stiffness. Next step was a multi-cycle dynamic axial load test of 4000 cycles under load control at 40 N/s. As before, the maximum load was set at 200 N and the minimum load was set at 20 N.This was followed by a pull-out test of the pin pairs from the cylinder segments. For this purpose, a multiplex board with a cut-out was screwed to the base plate. Each cylinder was placed in the indentation and secured to the base plate with four tension straps, for a total of 18 specimens. A cable wire attached to the input platen was looped around the two corresponding fixator couplings before a tensile force was applied at a speed of 50 mm/min until construct failure . Force aA regression line was calculated from each of the five load–displacement curves taken from the actuator of the testing machine before cyclic loading using Microsoft Excel 365 . The slope of this line represented the mean stiffness of the corresponding specimen. The multicycle load/displacement curves were inspected for irregularities and the constructs were manually examined for signs of damage and pin loosening. The ultimate load at failure was defined as the critical figure of the load/distension curves measured by the transducer of the testing machine resulting from the pull-out test.Statistical analysis was performed using SPSS Statistics version 27 . The mean stiffness of the assemblies was grouped  for one-way analysis of variance (ANOVA) followed by a post hoc Tukey test to detect significant differences between groups. The ultimate load at failure during the pull-out test was also grouped for a one-way analysis of variance (ANOVA) and followed by a post hoc Tukey test.The purpose of this biomechanical study was to demonstrate the effect of pin angulation in the monolateral fixator using a composite cylinder model. To this end, we established a test system to perform detailed mechanical testing. The resulting model system, as described in the Visually, there was no pin deformation during axial loading. Cantilever bending of the fixator bar was evident in all settings along the pin flight direction, resulting in irregular fracture gap reduction. The bar bent the highest degree in the divergent pin configuration and the least in the convergent configuration .There was a significant difference in mean stiffness between the three groups, with the converging configuration being the stiffest (27.13 ± 0.67 N/mm) and the diverging configuration being the least stiff (17.71 ± 0.19 N/mm). The parallel mounting showed an intermediate stiffness (20.24 ± 0.24 N/mm). The results are shown in There were no obvious signs of fatigue failure in any of the specimens during cyclic axial loading, which would have shown up as gross irregularities in the graphs . In no cThe mean ultimate load at failure of the diverging pin configuration was significantly higher (3.47 ± 0.51 kN) than that of the converging pin configuration (2.84 ± 0.25 kN). A significant difference between the pull-out strength of the parallel pins (3.16 ± 0.24 kN) and other pins could not be confirmed. These results are shown in A negative correlation between excessive axial and shear motion on the fracture side and early fracture healing has been demonstrated in numerous studies ,9,25,26.There have been numerous biomechanical investigations of the stability of novel external fixation designs compared to traditional monolateral constructs ,30,31,32Previous biomechanical studies have focused on the design of the basic monolateral external fixators ,12,15. AOni et al. have biomechanically demonstrated the beneficial properties of pin angle offset in the transversal plane on fixator stability in a Shearer-type fixator . In addiWhile the effect of angular pin separation in the sagittal plane on the stiffness of ring-type external fixators has been biomechanically investigated with positive results , there hAs we suspected, such a converging pin configuration resulted in increased stiffness under axial loading compared to a parallel pin configuration. There was a 34% increase from a mean of 20.24 N/mm to a mean of 27.13 N/mm when the pin pairs were convergently angled. In contrast, the stiffness decreased by 13% to a mean of 17.71 N/mm when the pin pairs were divergent. The effect is even more pronounced when comparing the converging configuration to the diverging configuration: It was 53% stiffer on average.The absolute stiffness values are at the lower end of the very wide range of data previously found for monolateral fixators under axial loads ranging from 0.13 to 1157.80 N/mm ,30. CompCantilever bending of the fixator bar under axial load was evident in all specimens. Contrary to the literature ,16,35, tIt is likely that increasing the frame offset and using a thicker or second bar would change the proportions in such a way that the pin stress would become more pronounced. We believe that such a condition would still favor the converging pin configuration. The bone fixator construct shown with its opposed angled pin arrangement, is reminiscent of a truss and would presumably best absorb the bending moment occurring in the pin flight direction under axial compression.In the case of parallel and convergent pin mounting, the asymmetric couplings were installed on the inside of the pin pairs. Due to lack of space between the pins in the diverging setup, the fixator couplings were placed on the outside of the pin pairs. If the couplings were mounted in the same way as in the parallel and convergent constellations, an even greater effect towards loss of stability can be expected.It has been suggested that the ideal positioning of a monolateral fixator on the tibia is with its pins set in anterior–posterior (AP) flight, as this is the best way to control the predominant bending moment that occurs in AP direction both under early mobilization and progressive weight-bearing ,16,36. AThe manipulation of the pin–bone interface by overdrilling 0.5 mm beyond the core diameter of the half-pins and the use of a two-way insertion technique must be critically mentioned. This procedure was chosen because a pretest without manipulation of the pin–bone interface did not show the hoped-for pin loosening after cyclic loading. The manipulation of the pin–bone interface could be expected to affect the primary stiffness of the constructs. No palpable loosening of the pins was observed either before or after cyclic loading. Even if the condition had an influence on the stiffness of the constructs, it was the same for all groups and thus not tangential to the relative stiffness.No fatigue failure occurred in either configuration during 4000 cycles of cyclic axial loading. We had postulated a superior pull-out strength of angled pin pairs over parallel pairs. Motivated through an assumed cant effect of angled pins independent of their threading into the bone, we aimed to induce pin loosening. This should have served as an amplifier of a supposed advantage in pull-out strength. The desired pin loosening was not achieved. Therefore, only the pull-out strength of a sound pin–bone interface was analyzed.Pin loosening relies upon adverse cortical bone remodeling and bone thread resorption as a reaction to shear forces at the pin–bone interface ,37. In aThere was a significant difference in pull-out strength between the diverging and parallel configurations, favoring the diverging configuration. This result could be explained by the possibility of a more even distribution of the tensile force exerted on the two fixator clamps by the cable wire looped around them. Because the clamps were closer together in the diverging configuration, the tensile force vectors of the cable were less angled to each other than in the parallel and even less than in the converging arrangement. Presumably, this resulted in a less pronounced preference of one pin over the other at the moment of pin slippage.A correlation between the stiffness of a fixator–bone construct and its durability at the pin–bone site has been suggested ,36. WhenThis study has several limitations. Due to reduced technical feasibility, cantilever and torsional testing were omitted and only axial loading experiments were performed. Because this loading mode best simulates the hypothesized predominantly compressive physiologic loading at the fracture site during weight-bearing ,39,40, aAnother weakness of this study is the use of previously clinically used fixator components. In addition, the material was reused throughout the different tests as long as no obvious plastic deformation had occurred.In addition to the aforementioned attempt to induce pin loosening in vitro, the question of the transferability of our pin-pair extraction technique to clinical reality remains. Applying equal tensile forces to two adjacent pins is unlikely to occur in vivo. The result of such an extraction test is highly dependent on the experimental setup, which allows for more or less distribution of the tensile force on the pins. Measuring and comparing the insertion and extraction forces of each pin, as performed by Pettine et al., and subsequent studies investigating the effect of pin coating with hydroxyapatite or calcium titanate ,23,24, wThe main strength of this study is its simple design. It has the character of a pilot study and, since its design conforms to ASTM, it is easy to reproduce with the possibility of modifying other parameters.The advantages of a stiffer construct as provided by a converging pin configuration are well known: better ability to maintain primary reduction of a fracture, better patient comfort due to less motion and therefore pain at the fracture level, higher chance of fracture healing, lower risk of pin loosening and, therefore, pin tract infection. All of this would be possible without sacrificing any of the benefits of the pin-to-bar-clamp external fixator. Compared to more dimensional or ring fixators, it is easier and faster to apply, limits the risk of iatrogenic soft tissue and nerve/vascular damage, and is less bulky and therefore more comfortable for the patient. However, due to its limited stiffness, it is rarely used for definitive fracture treatment. In locations where more dimensional or transfixation constructs are problematic due to limited pin corridors and conflicts with free extremity motion, as in the femur and upper extremity, this mounting option should be of great interest. In the case of fractures extending to the metaphyseal zone, joint integration by attaching a frame to the adjacent bone could be avoided with this method. In the femur, a forced wide pin offset could be counteracted by this pin angulation technique. Overall, the surgeon should be aware of this feature. However, to be clear, this study needs to be further validated and does not yet allow for definitive conclusions to actually change external fixation protocols in the clinic. In addition, our findings may open up new areas for product development, such as drill guides, multi-pin clamps, or entire scaffolds.In contrast, the negative effect of a divergent pin cluster arrangement on axial stiffness could be demonstrated. Knowing the weakening effect, this mounting option should be chosen with caution, as in the case of forced adaptation to soft tissue injuries.This is the first attempt to show the biomechanical effect of sagittally oriented pin angulation in a monolateral fixator. The mechanical behavior of constructs with further modified parameters under different loading modes needs to be evaluated in future studies. An alternative approach could be finite element analysis, which offers the possibility of targeting specific parameters with less costly and time-consuming methods.