This study evaluated the effect of UV absorbers on the dimensional accuracy of zirconia specimens fabricated by additive manufacturing using a digital light process. Zirconia suspension for additive manufacturing was prepared by setting the volume fractions  of various UV absorbers. The effect of UV absorber content was evaluated through curing thickness, geometric overgrowth model design, linear deviation, and microstructure evaluation before and after sintering. Statistical analysis was performed by Kruskal–Wallis H and post-tested by the Bonferroni correction method. There was no significant difference in the cure depth according to the presence or absence of the UV absorber, the difference in geometric overgrowth was from 2.1 to 12.5%, and the overgrowth significantly decreased as the amount of added UV absorber increased. This result may contribute to improved precision of 3D multilayer ceramic products. With the development of digital dentistry, computer-aided design/computer-aided manufacturing (CAD/CAM) system zirconia prostheses have gained attention for satisfactory esthetics, high biocompatibility, and improved mechanical properties compared to existing dental ceramics .Zirconia ceramic also has a variety of different applications other than dental implants and frameworks ,3,4, sucCurrently, zirconia prostheses are fabricated by subtractive manufacturing using a CAD/CAM system. A completely sintered zirconia prosthesis is obtained by milling and sintering a pre-sintered zirconia block. Studies show that 15–30% (approximately 20%) of linear shrinkage occurs due to sintering . HoweverThree-dimensional printing techniques (or additive manufacturing) have gained popularity in various fields, such as temporary prostheses, splints, and model manufacturing, due to the ability to consume less material than subtractive manufacturing and reproduce complex structures ,17,18. WThe photopolymerization method shows relatively higher precision, faster manufacturing speed, and higher surface roughness among these techniques. Therefore, it is more likely to be used in manufacturing dental prostheses . The DLPIn dentistry, strength and precision are enormous challenges when manufacturing zirconia prostheses by DLP additive manufacturing. The process requires a zirconia photopolymer suspension, generally including zirconia powder, photosensitive resin, photoinitiator, and dispersant. For zirconia to have high strength, a high zirconia volume fraction is required, creating difficulty in obtaining an appropriate degree of dispersion and viscosity . After mGeometrical overgrowth was introduced as a factor affecting manufacturing accuracy using DLP additive manufacturing zirconia ,30. GeomAdditive manufacturing using commercially available photopolymer resin for 3D printing not only controls but also compensates for the curing contraction of the resin and has shown clinically acceptable precision for manufacturing crowns and other prostheses . It is nZirconia photopolymer suspension was prepared based on the acrylate series commercialized with zirconia powder , and n = 5) [n = 5) .n = 5).Geometrical overgrowth occurs by curing a specimen into a wider area than the existing area exposed to the light source resulting from the light scattering effect of zirconia particles . This exn = 3) based on the volume fraction of the UV absorber, which was measured using digital vernier calipers (n = 3).Cure depth was measured to evaluate the effect of the UV absorber during lamination. Under the same conditions as the DLP 3D printer, a distance of 20 cm, a light of 100 mW/cm², and a light exposure time of 4 s were maintained. The conditions for degreasing and sintering were set based on the results of previous experiments . The micp < 0.0083 level. As normality was not satisfied in the Shapiro–Wilk test, the Kruskal–Wallis, a nonparametric test, was performed. The post hoc test was performed using the Bonferroni correction method.All statistical analyses were performed using SPSS 21.0 . All results were tested for significance at the It is essential to evaluate the strength and precision of additive manufacturing of zirconia dental prostheses using DLP. Geometric overgrowth, caused by over-curing resulting from the light scattering effect, is a factor that can affect precision. Light scattering is affected by the volume fraction of zirconia, the difference in refractive index between the zirconia powder and the medium, and the curing time ,29,30. Jp < 0.0083). No significant difference was observed based on the UV absorber ratio.The results highlight that the group prepared by adding 0.05% and 0.1% UV absorber to the zirconia suspension  showed a significantly greater degree of overgrowth in length and area than the group without the addition of UV absorber (Group O). Our previous study  measuredThe results show that the geometrical overgrowth was the lowest in the A100 group, containing 0.1% UV absorber, with overgrowth rates of 2.1% and 8.5% in length and area, respectively. When the overgrowth rate of the length was lower, the overgrowth was not constant by region, and the periphery of the rectangle tended to be rounded. Thus, we assumed that there was a difference in the area when the length was measured at a certain intermediate position. Additional studies are needed on the degree of overgrowth and the direction and pattern of overgrowth according to the specimen shape for precise additive manufacturing of zirconia.Zirconia prostheses are generally manufactured by setting the layer thickness between 25 and 100 μm during DLP additive processing . The pren = 3). Surface defects and microstructures were analyzed using a field emission scanning electron microscope . The specimens were sintered at 1450 °C to observe the surface after sintering.In this study, the thickness of the laminated layer was set to 100 µm and measured to check whether adding a UV absorber affects layering. 2 in group O. There was no significant difference in the cure depth in the groups with and without the UV absorber. Cure depths of 100 μm or more were observed in all groups. No surface defects or laminated zirconia layers were observed in the specimen after sintering in all groups. Thus, the geometrical overgrowth of pre-sintered bodies decreased when the UV absorber was added to the DLP-based additive manufacturing of zirconia, suggesting the possibility of increased accuracy of additive manufacturing of zirconia. Thus, sintering shrinkage compensation, reflecting geometrical overgrowth, is necessary to increase the accuracy in the additive manufacturing of zirconia.This study investigated the degree of geometrical overgrowth when a UV absorber was added to the additive manufacturing of zirconia using DLP. When evaluating the overgrowth rate, the length measurement ranged from 2.1% to 12.5%, and the area measurement ranged from 8.5% to 12.25%. Results showed a significant difference in the overgrowth between the groups without the UV absorber (Group O) and the group with 0.05% and 0.1% (A50 group and A100 group). The degree of overgrowth was the smallest in the A100 group and the highest in the O group. Length measurement revealed an average value of 40 μm in the A100 group and 251 μm in the O group. The average area was 0.490 mm


The composite gels of konjac glucomannan (KGM) and corn starch (CS) were prepared and treated by the freeze–thaw method. For KGM–CS gels, as the starch ratio rose from 0 to 100%, storage modulus (G′) decreased by 97.7% (from 3875.69 Pa to 87.72 Pa), degradation temperature decreased from 313.32 °C to 293.95 °C, and crystallinity decreased by 16.7%. For F–KGM–CS gels, G′ decreased by 99.0% , degradation temperature increased from 289.68 °C to 298.07 °C, and crystallinity decreased by 17.1% with more starch content. The peak in infrared spectroscopy shifted to a higher wavenumber with more starch and to a lower wavenumber by freezing the corresponding composite gels. The detected retrogradation of the composite gels appeared for KGM–CS with 80% starch and F–KGM–CS with 40% starch. The endothermic enthalpy of free water rose by 10.6% and 10.1% with the increase in starch for KGM–CS and F–KGM–CS, respectively. The results of moisture distribution found that bound water migrated to free water and the water-binding capacity reduced with more starch. The results demonstrated that the molecular interaction in composite gels was weakened by starch and strengthened by freezing. Amorphophallus konjac C. Koch. It has been widely used in food, chemical, pharmaceutical, cosmetic, coating, and painting industries [Konjac glucomannan (KGM) is a natural polysaccharide extracted from the tuber of dustries ,2. KGM idustries . It is wdustries ,5. In thdustries . The soldustries ,7. Compadustries ,9. Thus,dustries . KGM is dustries , carragedustries , and xandustries , to imprStarch is one of the most important and abundant polysaccharides in nature and is usually used to prepare gel, due to its wide distribution, green production, and low cost. The starch gel is formed via the damage of starch granules due to the rearrangement of starch molecules by hydrogen bonds . The proFreeze–thaw is an important technique to induce more interactions among biopolymer molecules and results in a denser and more aggregated network for hydrogels . Iijima This paper investigates the effect of different KGM/starch ratios and freeze–thaw treatments on the properties of the composite gels. The results could be used for optimal performance of the KGM–starch gels.The stable curve of G′ with increased frequency indicated that the frequency had no significant effect on KGM–CS and F–KGM–CS gels a,b and tThe G′ increased when the gels occupied by KGM (KGM100–CS0 and KGM80–CS20) were heated to about 70 °C c, which The G′ of the gels was promoted by freeze–thaw treatment accordingly d. In theAt small strains, the stable storage modulus means that the inner structure of the composite gel was fully developed e,f. The The yield strains of F–KGM–CS gels f decreasThermal gravimetric analysis is widely used to determine the thermal stability of materials . The weiThe degradation temperatures of KGM–CS gels were 313.32 ± 0.35 °C (KGM100–CS0), 305.77 ± 0.26 °C (KGM80–CS20), 299.45 ± 0.43 °C (KGM60–CS40), 294.88 ± 0.19 °C (KGM40–CS60), 293.95 ± 0.16 °C (KGM20–CS80), and 298.41 ± 0.39 °C (KGM0–CS100), respectively c. The reFor KGM–CS gels, more starch caused lower degradation temperatures and higher weight loss ratios, which suggested that starch was easy to dissociate compared to KGM. For F–KGM–CS gels, more starch caused higher both degradation temperatures and weight loss ratios, which suggested that the interaction between KGM and CS was promoted by the freeze–thaw treatment. Both the degradation temperature and weight loss ratio of F–KGM–CS gels were lower that of KGM–CS gels, which indicated that the composite gels became thermolabile due to some destruction of the network arising from the freeze–thaw treatment, while the interaction between the components was enhanced by freezing.Retrogradation is a common phenomenon in starch paste, which can be detected by DSC. The detected retrogradation was strengthened for KGM20–CS80 and KGM0–CS100 by the freezing, as both the peak temperatures and endothermic enthalpies of their frozen gels increased. In addition, in F–KGM–CS gels, the enhancement was promoted with more starch content. The results were consistent with the temperature sweep and TGA, in that freezing accelerated the interaction by physical crosslinking and hydrogen bonds in the junction zones of the polymeric network . For botThe composite gels exhibited a broad diffuse scattering at about 20° (2θ), and there were distinctions in the position and strength of the diffuse scattering . There w−1 in the spectrum of native KGM [−1 and 1062 cm−1 represented the stretching vibration of C–O–C groups. The peaks at 1383 cm−1 and 2923 cm−1 were from the –CH2– groups [−1 and 874 cm−1 assigned to the mannose of KGM weakened and disappeared when the starch ratio reached 100%. The absorption peaks at 761 cm−1, 850 cm−1, and 932 cm−1 assigned to the α–configuration of glycosidic linkage in starch were strengthened with more starch in the composite gels.There is a peak acetyl group at 1730 cmtive KGM , which d– groups ,43. The −1 to 3436 cm−1 in KGM–CS gels  free water with a melting temperature at 0 °C, (2) freezable bound water with a melting temperature at lower than 0 °C, and (3) non-freezable bound water, which did not crystallize even at −40 °C . The exi2 < 100 ms), including T21 (the peak time of relaxation time below 10 ms), T22 (the peak time of relaxation time between 10 ms and 100 ms), and T23 (the peak time around 100 ms) [21) and free water (T23) , weakly bound water (T22) and free water (T23)  appeared in the F–KGM–CS gels. As the starch ratio increased, the peak area of T22 reduced and its relaxation time rose. The implication that bound water migrated to free water by starch through the weakly bound water transition was consistent with unfrozen gels.Moreover, the LF–NMR analysis was further carried out to study the moisture distribution. According to the weakly bound water (1 ms < T 100 ms) ,47,48, ter (T23) a. The prer (T23) b. The T2The results of both DSC and LF–NMR analysis concluded that the free water in gels rose and the water-binding capacity reduced as starch ratio increased. More free water weakened the formation of strong intermolecular hydrogen bonds . This coThe rheological and thermal properties, crystallinity, and moisture distribution of the composite gels with or without the freeze–thaw treatment were analyzed to explore the effect of the ratio of KGM and CS and freezing on gelling behavior. The results demonstrated that the molecular interaction of composite gels was inhibited by starch and strengthened by freezing. This study will be a driving force to understand the influences of KGM, starch, and freezing on the composite gels, which are beneficial to develop gel functions and reduce costs, such as in low-calorie foods, meat alternatives, and the encapsulation and delivery of aroma compounds.Native KGM flour  was purchased from Konson Konjac Science and Technology Co., Ltd. , and corn starch (CS) (31.95% of amylose content) was from Huifeng biotechnology Co., Ltd. . 3,5–DNS, formic acid, glucose, phenolsodium hydroxide, sodium hydrogen sulfite, seignette salt, and sulfuric acid were purchased from Kermel Chemical Reagent Co., Ltd. .Starch was precisely weighed and dispersed in alkali solution  at 25 °C by magnetic stirring (150 rpm). Starch slurry was heated (95 °C) in water bath for 90 s and then cooled to 25 °C. KGM flour was added slowly to starch solution. The mixture was kept stirring and heated at 80 °C for 80 min, followed by standing at 25 °C for 24 h to stabilize the KGM–CS gels. A series of composite gels were obtained by changing KGM/CS ratio: 100/0, 80/20, 60/40, 40/60, 20/80, and 0/100 . The composite gels were frozen (−18 °C) for 24 h, then thawed at 25 °C for 2 h to obtain the F–KGM–CS gels. Accordingly, they were marked as F–KGM100–CS0, F–KGM80–CS20, F–KGM60–CS40, F–KGM40–CS60, F–KGM20–CS80, and F–KGM0–CS100.The rheological measurement was performed using controlled strain rheometer  with a parallel plate . Temperature was controlled by peltier steel plate. The samples were covered with silicone oil to prevent water evaporation. The linear viscoelastic region of composite gels was determined by the amplitude sweep measurement from 0.1% to 50% with constant frequency (1 Hz) at 25 °C. After amplitude sweep, 1% strain was chosen for further examination. The frequency sweep was obtained from 0.1 Hz to 20 Hz and temperature sweep was conducted from 25 °C to 95 °C with a heating rate of 5 °C/min.TGA measurement of composite gels was carried out by TA Q50 apparatus . Lyophilized sample (10 mg) was weighed and deposited in the platinum basket. The sample was heated from 40 °C to 600 °C with a heating rate of 10 °C/min under nitrogen atmosphere at a constant flow rate of 40 mL/min.DSC measurement was carried out with TA Analysis software . The system was calibrated with pure indium. TA Tzero aluminum pans were used, and an empty pan was the reference. The samples (10 mg) were cooled to −40 °C and then heated to 40 °C at a rate of 5 °C/min with the gas (N2) at 40 mL/min to measure the free water content in composite gels. In addition, the samples were heated from 40 °C to 200 °C to measure retrogradation of composite gels.The crystallinity of the powder of the composite gels was carried out by D8 DVANCE . Samples were analyzed from 2θ of 5° to 45° with a scanning rate of 4°/min. The experiment condition was 0.02° of step size, 40 kV of voltage, and 30 mA of current.−1 to 4000 cm−1 by FT–IR spectrometer . The dried KBr and 1% samples were mixed to prepare discs for FT–IR analysis.Composite gels were lyophilized and ground to powder. FT–IR analysis was carried out from 400 cm2) were as follows: 4000 ms of waiting time, 0.2 ms of echo time, 18,000 echo numbers, and 16 scan numbers.Moisture distributions of the composite gels were measured by LF–NMR analyzer  under Carr–Purcell–Meiboom–Gill (CPMG) sequences. Approximately 0.5 g of composite gel was transferred to a 10 mm diameter NMR glass tube. The measured parameters of transverse relaxation time  was processed using SPSS 24.0 .


Cervicogenic headache (CEH) is a secondary headache caused by lesions of the cervical spine and surrounding soft tissues. Cervical muscle dysfunction may be related to the onset of CEH. However, whether cervical muscle stiffness changes in patients with CEH has not been well studied. The purpose of this study was to explore changes in superficial cervical extensor muscle stiffness in patients with CEH using shear wave elastography (SWE).In this study, 19 patients with CEH and 20 healthy controls were recruited. Superficial cervical extensor muscle stiffness was obtained from SWE, and the SuperLinear SL10-2 MHz linear array probe in the musculoskeletal muscle mode was chosen as the transducer. Regions of interest in the trapezius (TRAP), splenius capitis (SPL), semispinalis capitis (SCap), and semispinalis cervicis (SCer) were manually segmented. Correlations between superficial cervical extensor muscle stiffness and visual analog scale (VAS) scores, age, and body mass index (BMI) were analyzed using Pearson's correlation. Receiver operating characteristic (ROC) curve was used to investigate the diagnostic ability of superficial cervical extensor stiffness for CEH.p < 0.05). Increased stiffness was also observed in SCer on the non-headache side of patients with CEH compared to healthy controls (p < 0.01). In patients with CEH, SCer stiffness was positively correlated with VAS scores , but no correlation was found between other muscles and VAS scores (p > 0.05). The areas under the curve of TRAP, SPL, SCap, and SCer in diagnosing CEH were 0.766, 0.759, 0.964, and 1.000, respectively.Superficial cervical extensor muscle stiffness on the headache side of patients with CEH was higher than that on the non-headache side and in healthy controls (Increased stiffness was observed in the superficial cervical extensor muscles on the headache side of patients with CEH. SCer stiffness was correlated with headache intensity in patients with CEH and may provide clues for the diagnosis of CEH. Cervicogenic headache (CEH) is a common secondary headache caused by lesions of the cervical spine and surrounding soft tissues. The prevalence of CEH has been reported to be 0.4–2.5% in the general population  and as hLesions of the cervical extensor muscles may be related to the onset of CEH. The cervical extensor muscles are the basis for maintaining normal function of the cervical vertebra. The cervical extensor muscles include the trapezius (TRAP), splenius capitis (SPL), SCap, semispinalis cervicis (SCer), and multifidus  from theShear wave elastography (SWE) is a non-invasive, quantitative, and real-time imaging of soft tissue stiffness, which is considered to be more objective and reproducible than compression sonoelastography . SWE useIn this study, SWE was used to study superficial cervical extensor muscle stiffness in patients with CEH and in healthy controls. The shear elastic modulus of TRAP, SPL, SCap, and SCer was quantitatively measured. In addition, we investigated the factors that may be associated with superficial cervical extensor muscle stiffness and explored the diagnostic ability of superficial cervical extensor stiffness for CEH.A total of 19 patients with unilateral CEH were recruited for this study from the Department of Rehabilitation Medicine of Weifang People's Hospital between June 2021 and October 2021, and another 20 healthy volunteers with matched age and gender were recruited as a control group. Patients with CEH were diagnosed by a neurologist (SYZ) with more than 30 years of experience. This study was approved by the Local Ethics Committee, and all patients were fully explained about this study and were given informed consent.Inclusion criteria for patients with CEH were based on the International Classification of Headache Disorders-3 (ICHD-3). These criteria included clinical or imaging evidence of a lesion within the cervical spine or soft tissues of the neck, known to cause headache. Any headache fulfilling the following standards or evidence of causation was demonstrated by at least two of the following: firstly, the headache has a temporal relationship to the onset of the cervical disorder; secondly, the headache can be significantly relieved as the cervical spondylosis improves or subsides; thirdly, the cervical range of motion is reduced and the headache is significantly worsened by provocative maneuvers; and fourthly, the headache is abolished following diagnostic blockade of a cervical structure or its nerve supply. Patients with CEH aged 16–55 years without cognitive impairment were included in this study. Exclusion criteria included combination with other types of headaches, a previous history of severe head and cervical trauma or cervical surgery, cervical congenital malformation, osteoporosis, cervical vertebra fracture, bone destruction, patients with mental or psychological disorders unable to cooperate with the examination, pregnant women, and patients after treatment such as massage, physiotherapy, and muscle relaxants.Inclusion criteria for healthy controls were 16–55 years of age, with no history of CEH and cognitive impairment. Exclusion criteria included other types of headaches, a previous history of severe head and cervical trauma or cervical surgery, cervical congenital malformation, and pregnancy.Before measuring stiffness, we measured the headache intensity with the visual analog scale (VAS)  in the CThe subjects were lying prone, with their shoulders in abduction and external rotation, hands overlapped under the forehead and the chest supported by a pillow to make the neck in a neutral position. The stiffness of the muscles on the left and right sides of the back of each subject's neck was measured. Muscle stiffness was measured by shear wave ultrasound elastography , and a SL10-2 MHz linear array probe in the musculoskeletal muscle mode was chosen as the transducer, with a shear elastic modulus up to 180 kPa. Ultrasound was performed by a sonologist (YNY) who had 5 years of experience in musculoskeletal ultrasound and was blinded to the information of subjects. The measured shear elastic modulus values were expressed in kPa, the larger the shear elastic modulus value, and the greater the muscle stiffness.v2, where ρ is the muscle density and v is the speed of the shear wave propagating in the muscle tissue . Independent samples t-test was used to compare age and BMI between patients with CEH and healthy controls. Correlations between cervical extensor muscle stiffness and VAS scores, age, and body mass index (BMI) were analyzed using Pearson's correlation. The diagnostic ability of the stiffness of TRAP, SPL, SCap, and SCer for CEH were determined by performing receiver operating characteristic (ROC) curve analysis. A two-tailed p < 0.05 was considered significant.In this study, all statistics were performed with Statistical Product and Service Solutions . Numerical data were presented as mean ± standard deviation (SD), and the Shapiro–Wilk test was used for normality test. One-way analysis of variance (ANOVA) was used for comparison of muscle stiffness between the different groups, and the least significant difference (LSD) test was used for p = 0.227) and BMI (p = 0.888). The mean VAS score in patients with CEH was 5.21 ± 1.13 (range 3–7)  and 20 age-matched healthy controls (10 men and 10 women) were included in the analysis. The mean age of patients with CEH and healthy controls was 27.47 ± 8.38 (range 16–44) years and 24.60 ± 5.92 (range 16–40) years, respectively. The mean BMI of patients with CEH and healthy controls was 21.75 ± 2.65 (range 17.3–26.8) and 21.63 ± 2.74 (range 17.6–25.8), respectively. There were no statistically differences between the two groups for age (nge 3–7) .p < 0.001). Compared to healthy controls, the stiffness of TRAP, SPL, SCap, and SCer was significantly increased in the headache side of patients with CEH (p < 0.01). Significantly increased stiffness in SCer was found on the non-headache side of patients with CEH compared to healthy controls (p < 0.01)  . In addir = 0.481, p = 0.037). No correlation was found between stiffness and VAS in TRAP , SPL , or SCap  , SPL , SCap , or SCer . No correlation was found between stiffness and BMI in TRAP , SPL , SCap , or SCer . Similarly, no correlation was found between superficial cervical extensor muscle stiffness on the non-headache side and VAS, age, or BMI.In patients with CEH, headache side stiffness and VAS scores were positively correlated in SCer (= 0.455) . There wThe ROC curve analysis showed that the area under the curve (AUC) of TRAP, SPL, SCap, and SCer were 0.766, 0.759, 0.964, and 1.000, respectively . The senIn this cross-sectional study, we found that the stiffness of all the examined superficial cervical extensor muscles was higher on the headache side of patients with CEH than on the non-headache side and in healthy controls. The stiffness of SCer on the non-headache side of patients with CEH was also increased compared to healthy controls. In patients with CEH, the stiffness of SCer was positively correlated with VAS scores. There was no correlation between the stiffness of lateral TRAP, SPL, SCap, SCer, and age or BMI. Measured muscle stiffness had fair combined sensitivity and specificity in the diagnosis of CEH, and the stiffness of SCer had the best diagnostic ability to distinguish patients with CEH from healthy controls. The results were consistent with the manifestations of cervical lesions and their soft tissues in patients with CEH. Our study provided evidence that superficial cervical extensor muscle stiffness was increased in patients with CEH, with data supporting the clinical findings of increased cervical muscle stiffness in patients with CEH.In vitro animal studies showed that type I fibers were harder than type II fibers  and migraine, making them difficult to distinguish. It has been found that the stiffness of TRAP, SPL, SCap is significantly greater in patients with TTH and migraine than in healthy controls , 43, butOur study has several limitations. Firstly, although SWE is performed at a specific vertebra level and in a standard position from the spinous process, intramuscular variations may make the data to have a small extent of variability , 44. SecIn conclusion, our study found significantly increased stiffness in the superficial cervical extensor muscles of patients with CEH. The stiffness of the superficial cervical extensor muscles, especially SCer, has a fair diagnostic ability for CEH. Whether the stiffness of the superficial cervical extensor muscles is related to the severity of CEH remains to be studied.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Medical Ethics Committee of Weifang People's Hospital. The patients/participants provided their written informed consent to participate in this study.LZL, YNY, SYZ, XZY, and JCF developed the study concept and design. LZL, YNY, and JCF collected clinical and imaging data and analyzed and interpreted the data. PWG, CFX, and XG analyzed the data. SYZ and XZY designed and revised this manuscript. LZL wrote the first draft. All authors critically reviewed this manuscript, contributed to this article, and approved the submitted version.This study was supported by grants from the Weifang Health Commission Fund Project (wfwsjk-2021–161).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.


Correction to: Journal of Clinical Immunology10.1007/s10875-022-01298-2The article note is incorrect. It should have been stated as “Alfonso José Tapiz i Reula, Alexis-Virgil Cochino, Andreia L. Martins and Diego Angosto-Bazarra contributed equally to this work.”The original version has been corrected.


Correction to: Journal of Clinical Immunology (2021) 41:1633–164710.1007/s10875-021-01098-0ADA2 for patient 26 (P26) is p.Leu188Pro, and not p.Lys188Pro, as reported in Table 2.This erratum is to clarify that the allele 1 of


The pathophysiological process of ischemia and reperfusion injury (IRI), an inevitable step in organ transplantation, causes important biochemical and structural changes that can result in serious organ damage. IRI is relevant for early graft dysfunction and graft survival. Today, in a global context of organ shortages, most organs come from extended criteria donors (ECDs), which are more sensitive to IRI. The main objective of organ preservation solutions is to protect against IRI through the application of specific, nonphysiological components, under conditions of no blood or oxygen, and then under conditions of metabolic reduction by hypothermia. The composition of hypothermic solutions includes osmotic and oncotic buffering components, and they are intracellular (rich in potassium) or extracellular (rich in sodium). However, above all, they all contain the same type of components intended to protect against IRI, such as glutathione, adenosine and allopurinol. These components have not changed for more than 30 years, even though our knowledge of IRI, and much of the relevant literature, questions their stability or efficacy. In addition, several pharmacological molecules have been the subjects of preclinical studies to optimize this protection. Among them, trimetazidine, tacrolimus and carvedilol have shown the most benefits. In fact, these drugs are already in clinical use, and it is a question of repositioning them for this novel use, without additional risk. This new strategy of including them would allow us to shift from cold storage solutions to cold preservation solutions including multitarget pharmacological components, offering protection against IRI and thus protecting today’s more vulnerable organs. Nowadays, organ transplantation provides the best available solution and is the clinically accepted treatment for end-stage organ failure including end-stage renal disease (ESRD) and liver failure . In thisIRI has been shown to be an important contributor to early graft dysfunction (EGD), specifically to renal delayed graft function and early allograft dysfunction in liver ,4,5,6. IThe currently accepted standard method for the preservation of allografts worldwide is cold storage. This method has the enormous benefit of simplicity. However, it involves a phase of ischemia, since from organ procurement, during transport and until transplantation into the recipient, the organ is inevitably deprived of blood, which otherwise would provide oxygen and nutrients. To protect an organ during this phase, its metabolism is slowed by flushing and conditioning it with a cold solution at +4 °C, called a cold storage solution (CSS), and it is transported in a hypothermic box. Recently, perfusion machines have appeared on the market, which replace the simple hypothermic box and perfuse the organ with an organ preservation solution at +4 °C, in this case called machine perfusion solution (MPS). Both techniques are used in clinical practice, and in both cases, the organ is in contact with a cold solution ,16,17,18After the ischemic phase, the organ is rinsed with a physiological solution, or blood, prior to implantation in the recipient. Graft damage occurs gradually during ischemia with further deterioration taking place during reperfusion after revascularization. During the organ preservation process, the first level of protection is provided by hypothermia, since this reduces the metabolic requirements of the graft.The solutions used for cold storage or machine perfusion are the vectors by which hypothermia is applied. Indeed, their composition plays a central role in optimizing the quality of the preserved organs and inhibiting or preventing IRI.In 1988, Belzer et al. defined the concept of abdominal cold storage solution for kidneys, liver and pancreas and protection against IRI -3-[(2-methoxyphenoxyethyl)amino]-2-propanol], is a nonselective β-adrenergic antagonist, which exerts a vasodilatory effect via the blockage of α1-adrenergic receptors . Apart f3+ ions [2− anions and OH·radicals from both aqueous media and membrane lipids after reperfusion of ischemic myocardium [Carvedilol-induced inhibition of lipid peroxidation has been attributed to its capacity to sequester Fe3+ ions ,126. Mor3+ ions ,130,131.ocardium ,130,132.ocardium  through ocardium . A recenocardium .2+ homeostasis by modulating Ca2+ intracellular concentration independently of phospholipase-C (PLC), thus avoiding mitochondrial dysfunction [Carvedilol improves Cafunction ,137. In function . Moreovefunction ,140,141.function ,139. Carfunction . Indeed,function . Several studies have indicated that carvedilol improves endothelial function . Indeed,Carvedilol, used as an additive to a preservation solution, is of interest due its potential to protect against IRI during transplantation. It has been widely reported that AMPK activation promotes NO synthesis, which in turn protects steatotic and nonsteatotic livers against IRI. Ben Mosbah et al. demonstrated a protective action of carvedilol when added to a UW solution ; they foOn the basis of the cited literature, these three molecules might offer a new strategy, and they may be capable of acting simultaneously, thereby regulating different targets responsible for IRI and thus protecting the organs. Transplantation is a complex procedure that requires optimal conditions to ensure the long-term survival of the graft. The inevitable storage period between removal of the organ from the donor up to transplantation to the recipient generates an IRI.Current hypothermic solutions have their advantages and limitations. However, they all contain the same protective agents, which have not proven to be effective in protecting against IRI.Moreover, the increase in organs from extended criteria donors, which are more sensitive to IRI, combined with a better understanding of impacts of IRI, calls for an even better attention to preservation.Thus, the current main goal in the field of organ preservation is to define a formulation that includes new stable pharmacological components that protect against IRI without involving additional clinical risks.Trimetazidine, carvedilol and tacrolimus, which are safe multitarget drugs, seem to be good candidates for inclusion in the formulation of a new improved solution; they could be included into an optimal extracellular-type washing solution base, rich in sodium and with PEG as an oncotic agent.This strategy is about to move from today’s cold storage solution to tomorrow’s cold preservation solution. It will design a potential route towards optimized organ protection in transplantation.


Worldwide, over 1,300 pediatric kidney transplantations are performed every year. Since the first transplantation in 1959, healthcare has evolved dramatically. Pre-emptive transplantations with grafts from living donors have become more common. Despite a subsequent improvement in graft survival, there are still challenges to face. This study attempts to summarize how our understanding of pediatric kidney transplantation has developed and improved since its beginnings, whilst also highlighting those areas where future research should concentrate in order to help resolve as yet unanswered questions. Existing literature was compared to our own data of 411 single-center pediatric kidney transplantations between 1968 and 2020, in order to find discrepancies and allow identification of future challenges. Important issues for future care are innovations in immunosuppressive medication, improving medication adherence, careful donor selection with regard to characteristics of both donor and recipient, improvement of surgical techniques and increased attention for lower urinary tract dysfunction and voiding behavior in all patients. The first successful pediatric kidney transplantation was performed in 1959 at the University of Oregon in Portland, USA . The fieBoth patient and graft survival improved dramatically; from a 1-year patient survival of 70% in 1970 into a current 1-year patient survival of 97% and 5-year graft survival of 89% –6. ConseMultiple factors led to improved graft survival and quality of life (QOL) in pediatric kidney recipients, for example new developments in immunosuppression protocols. Infections, both bacterial and viral, used to be responsible for high morbidity and mortality in the early years of transplantations , 9. SubsPediatric transplantation differs from adult transplantation as unique pediatric concerns related to development, growth, viral infections, congenital disorders and adherence need to be managed , 12. AwaDespite this increased survival rates, we aim to further optimize care for these patients. Considering the current allograft half-life of 12 to 15-years, most pediatric kidney recipients will require a re-transplantation during their lifetime .Moreover, since survival has increased, future research needs to focus on the long-term effects of kidney transplantation: maintaining QOL and minimizing the side effect of immunosuppressants. Due to relatively small numbers of transplantations per center, it takes time to gain expertise.This non-systematic review presents a summary of the current available literature. The aim was to present an overview of lessons learned during the last 5 decades of pediatric kidney transplantation and to identify unresolved fields waiting to be unraveled. Analysis of still existing lacunas in pediatric kidney transplantation care are essential to further optimize outcome.In addition we present our own results of 411 single center pediatric kidney recipients that were transplanted in our center in the time period 1968–2020.A comprehensive literature search was conducted in the databases PubMed, Cochrane, EMBASE and MEDLINE for relevant English-language articles. In addition we followed citations from the primary references to relevant articles that the databases could not locate. The search was based on the following MESH-terms: pediatric kidney transplantation, donor selection, donor age, living related and unrelated kidney donation, post mortal kidney donation, prognostic factors, dialysis, pre-emptive transplantation, immunosuppressive drugs, corticosteroid withdrawal, long-term outcome/ graft survival, rejection, infections, and surgical techniques, complications, including ureteroneocystostomy. All abstracts were screened for relevant articles. Full text relevant articles were reviewed and included.This article focused on pre-operative issues like donor selection, pre-emptive transplantation and screening for lower urinary tract dysfunction (LUTD). Besides this it covers peri-operative factors such as anastomosis technique and surgery for really small children. Eventually it describes post-operative factors like graft- and patient survival, immunosuppression and the need for transplantectomy.p < 0.05.In order to evaluate our own practice and to identify dissimilarities with previous research we compared outcomes retrieved from existing literature to outcomes of 411 single-center pediatric kidney recipients transplanted between 1968 and 2020 in our center. For this, population data were analyzed using SPSS Statistics 25.0 and Graphpad Prism 5.0. Differences were considered statistically significant at In this section we will present an overview of current literature on the most important (modifiable) factors in pediatric kidney transplantation (age 0–18 years) and highlight existing controversies that remain to be clarified.Since 1959, patient survival increased significantly. Whereas, 5-years patient survival was 91% before 1990 it improved to up to 98% after 2010, mainly due to decline in infections . AlthougAs infection rates decreased other long-term factors became more important like malignant diseases and cardiopulmonary complications. Both these complications are responsible for respectively 12 and 15% of current 5- and 10-year patient survival .In 1990, 5-year graft survival was ~77% for the living donations (LD) and 57% for the deceased donations (DD) . In thisNowadays, acute rejection rates have decreased to 10–15%, due to improvements in pre-operative donor selection, peri-operative management and immunosuppressive regimes. Although rates of acute rejection have immensely diminished, chronic rejection and acute rejection remain the leading causes of graft loss  . Other iIn our center, since 1968, 411 kidney transplantations have been performed in patient aging 0–18 years.Similar to the literature, patient survival increased from a 5-year survival of 93% in recipients transplanted before 1990 to 98% when transplanted after 2010. Overall, infection was the most important cause of mortality (25%), followed by cardiovascular complications and malignancy .p < 0.01)]. In general, LD resulted in better 5, 10, and 20 year graft survival compared to DD (p < 0.01). Majority of graft loss in our center was caused by both forms of rejection (75%). Other important causes were recurrence of primary disease (5%) and thrombosis (6%). Causes of graft loss did not change over time.Graft survival increased significantly over ascending era's even when stratified for DD/LD [Most pediatric kidney recipients are exposed to dialysis prior to their transplantation [51% to hemodialysis (HD) and 28% to peritoneal dialysis (PD)]. Current incidence of pre-emptive kidney transplantation (PKT) in children is 20% in Europe . HoweverIn theory, dialysis has several disadvantages for children suffering from ESKD. Dialysis is associated with negative effects on growth, anemia, bone mineral regulation and cardiovascular status due to chronic volume overload and uremic toxins –26. MoreDespite these theoretical objections to dialysis, literature showed conflicting results of PKT in children. Some studies report better graft and patient survival in PKT –30 whilsThere are several possible explanations for these conflicting results.Undervaluation of PKT might be due to the relatively short duration of dialysis in children compared to adults. Period of dialysis is thought to predict survival since a longer time on dialysis was associated with increased risk of adverse events , 26, 34.Amaral et al. showed significant graft survival benefits in pediatric recipients after PKT compared to those on dialysis for as little as 6 months . This waOvervaluation of PKT might be caused by selection bias. Recipients of PKT are more likely to be healthier, better nourished, have better residual kidney function and more likely to receive a graft from a LD compared to those on dialysis , 35.Another factor is lead time bias. The treatment of choice, in this case transplantation, was given at an earlier stage in PKT than after dialysis which results in a longer follow-up after transplantation. This can cause a perceived advantage in PKT, as the graft survival time is calculated from an earlier starting point than in post-dialysis transplantation .Additionally, a possible explanation for the conflicting results is the limited follow-up time. As the risks of dialysis are mainly cardiovascular disease, consequences are expected later in life and affect patient survival rather than graft survival. More long-term research could provide better answers.An important barrier to PKT is a lack of patient education as many patients and potential living donors are not aware of the possibilities of PKT , 38. BesIn our center, the rate of PKT increased over time, from 6% before 1990 to a current number of 46% . Median Donor selection is an important factor for graft survival: LD is considered to be preferable to DD since it results in both better graft and recipient survival , 44. HowLiving related donation allows proper HLA-matching and limitation of ischemia time. And using living (un)related donors allows paired exchange.In pediatric transplantation, LRD rates exceed those in the adult population since the donors are often the parents of the child.Previous studies stated that maternal donation might be preferable to paternal donation since it results in decreased rate of acute rejection in the youngest recipients (<4 years) , 46. ThiDespite an overall increase in LD in Europe, the numbers of patients on donor kidney waiting lists are stabilizing . ImpedimA possibility to expand the donor pool is the use of living unrelated donors. Graft outcome after LURD was shown to be superior to DD –54. In aAlthough ethics of organ donation have always been a sensitive issue, this might be of more importance in (unrelated) living donation . SeveralNon-commercial LURD is allowed in most Western Countries such as the Netherlands, following the recommendations of the Council of Europe .Previous research in adults has shown advanced donor age to result in poor graft survival , 59.This deleterious effect of high donor age seems less evident in pediatric recipients. Chesnaye et al. showed that the risk of graft failure in older living donors (50–75 years old) was similar to that of younger living donors . On the Conflicting results have been published concerning the effect of HLA-matching. Whereas, some studies showed superior results for children receiving a poorly HLA-matched LD kidney compared to a well-matched DD kidney , 63 mostHowever, the definitions of poor and well-matched donation differ between studies. Additionally, geography might play a role in this context as cold ischemia times would be increased if DD grafts need to travel large distances.Currently, the trade-off between time on a waiting list and HLA-mismatching remains unsolved and needs further exploration in the future.p = 0.01) even when stratified for decade of transplantation.In our center, the rate of LD and the donor age increased over time . Median In total, 3% of the kidneys were from a LURD and 33% from a LRD, mostly parents (18% father and 11% mothers). There was no significant difference in graft survival between maternal and paternal donors.p < 0.01).In addition, median number of HLA mismatches significantly increased over time  and poses surgical challenges in terms of size differences in body cavity and vessel diameters , 68. PreTrying to fit an adult-size graft in a small retroperitoneal space is challenging and might lead to increased abdominal pressure and impaired graft vascularization , 75. In In adults, the graft is commonly placed retroperitoneal whereas in children graft placement depends on the size of both the abdomen and the graft. In the youngest children , many centers use intra-abdominal placement and implant the graft on the inferior vena cava and aorta rather than on the more commonly used iliac vessels , 79, 80.Despite the advantage of larger space for the graft, intra-abdominal placement has some disadvantages including risk on bowel injury and a more difficult access for future interventions such as graft biopsies or PD.Previous studies showed small children (under 15 kg) to be at increased risk for thrombosis compared to older children (OR 0.11–0.85) , 81, 82.Additionally, very specific individual management might be needed in case of rare and complex associated medical conditions associated with prematurity, severe anatomical anomalies, mental illnesses, syndromal anomalies or extensive urological, or surgical previous procedures. This care should include a medical point of view of all related specialties, nursing care experts and psychological support.Despite the relatively high complication rates, graft- and patient survival in small children have improved in the last decades . Nowadays, outcomes of transplantation in infants are comparable to those in older children , 78, 83.Accurate imaging of recipients and potential donors allows health care providers to make well-educated choices regarding the favorable surgical technique and postoperative care.Although kidney transplantation is shown to be safe and successful in very small children, there is still some controversy concerning the optimal timing of the transplantation. On the one hand, early transplantation could avoid dialysis and allows better physical and neurological growth in these young children. On the other hand, early exposure to immunosuppressive therapy might result in severe infections and long-term side effects. Besides, with current graft survival, transplantation at a very young age implicates multiple re-transplantations in a life time, which are known for decreased graft survival compared to primary transplantations –86.Currently, there is no consensus on a minimal age or weight for transplantation. Whereas, some centers perform transplantations in children above 6 kg, others use a minimum age of 2 years , 72, 87.In our center, we started a program for transplantations in children weighting <20 kg in 2012. A multidisciplinary team including pediatric nephrologists, pediatric urologists, pediatric anesthesiologists, pediatric ICU specialists, vascular surgeons and pediatric surgeons as well as a paramedical team started this program with satisfying outcome .Special attention is paid to the disparity between the size of the kidney graft and the length of the recipient and therefor the size of the body cavity.Since the start of this special program in 2012, 13 children with a weight below 15 kg have been transplanted thus far . Up to this moment all recipients are alive with a functioning graft [median follow-up of 89 months (range 3–221)].p = 0.26)  .Urological causes for ESKD are seen in 25–40% of the pediatric kidney recipients and encompass mainly posterior urethral valves (PUV), vesico-ureteral reflux (VUR) and neurogenic bladder . NephrolLower urinary tract dysfunction (LUTD) might affect pediatric kidney graft outcome . LUTD isIn children with PUV, myogenic changes in the bladder wall result in abnormal contractility and different sensations in for example a full bladder. This ultimately might induce abnormal voiding behavior and high intra-vesical pressures. The normal bladder cycle is interrupted in patients with PUV and high pressure might cause fibrosis in the bladder walls. The prevalence and severity depends on the severity of previous mentioned changes in the bladder wall –99. TherThere are several factors that could contribute to development of LUTD in pediatric renal recipients without an urological history. Long pre-transplantation polyuria may lead to overdistension of the bladder which results in diminished sensation and therefore abnormal voiding patterns . On the At this moment, little information is available on the effect of immunosuppressive medication on the bladder wall .The general believe is to treat LUTD as much as possible before transplantation in order to protect the kidney graft from a high-pressure lower urinary tract . AlthougRegardless of the actual effect on graft function, LUTD in children results in diminished quality of life and increased morbidity, especially in the case of urinary incontinence –110.Optimal timing and screening methods remain to be determined. Multiple diagnostic tools are available including  frequency voiding charts (FVC), Urodynamic studies, Ultrasound (US), Voiding Cystourethrogram (VCUG), uroflowmetry (FR), and post voiding residual (PVR) measurement. Since most patients do not need invasive diagnostics, PVR was found to be the most accurate predictor for prognosis . HoweverUrotherapy, which is defined as non-surgical, non-pharmacological treatment for LUTD, is considered to be the cornerstone in the treatment in otherwise healthy children , 113. ItThe timing of bladder augmentation remains controversial. If indicated, bladder augmentation is mostly performed before transplantation . An advaThe need of pre-transplantation bladder cycling remains unclear, although several authors argument that this would improve outcome, it was not shown to be beneficial , 121.Another controversial topic is the treatment of asymptomatic bacteriuria (AB) which occurs in 17–51% of adult kidney recipients . MultiplThe role of bladder rinsing with hyaluronic acid and chondroitin sulfate is unclear. Several studies have shown beneficial effects in individual pediatric patients, but those were limited in the number of participants , 127.n = 89) of the 411 pediatric kidney recipients had an urological cause for their ESKD. In a prospective study of 56 patients, we screened all recipients for LUTD and treated them if indicated. LUTD was diagnosed in the majority of patients (71%) regardless of the underlying cause of kidney failure. This indicates that most pediatric transplant recipients do not have adequate voiding behavior, normal bladder capacity, and micturition frequency.In our center 22%  of 70 mmHg is recommended in adults, administration of excessive fluids or vasopressors might be harmful in children. Therefore, adjustment of the target MAP to the donors MAP and visual judgement of perfusion is favored in this population.Methods for managing hemodynamics are the administration of fluids and the use of vasoactive medication.Norepinephrine is recommended in patients that do not respond to fluid administration. It prevents post reperfusion hypotension which is commonly seen in small pediatric patients that receive a kidney form an adult donor , 131.In very young children (<5 years) hemodynamic challenges are even bigger. Because of large differences in vascular sizes between donor and recipient, renal arterial blood flow can be compromised. Additionally, a large kidney demands a persistent increase in cardiac output of the child in order to meet the flow demands of the graft. Therefore, close hemodynamic monitoring is of utmost importance in these patients and cardiac output measurement during anesthesia should be considered .In our center, anesthesia for children below 40 kg is done by dedicated pediatric anesthesiologists.2 and oximetry. In children <20 kg we use Pulse Contour Cardiac Output (PiCCO) technique for advanced hemodynamic monitoring , congenital anomalies, chronic infection, refractory hypertension, and malignancy .However, some of these indications are rather relative indications. Arguments against this procedure include the need of additional surgery and anesthetics, the risk of peritoneal laceration and the benefits of residual urine production.Theptimal timing of nephrectomy has to be determined as well. Whereas, nephrectomy was commonly conducted before transplantation, some authors are in favor for post-transplantation nephrectomy because of the benefits of pre-emptive transplantation, minimization of sensitization and better clinical condition . Other sCurrently, nephrectomy by means of a surgical intervention is the most common form of practice. However, various studies reported on alternatives like renal arterial embolization and medical nephrectomy by means of indomethacin or an ACE inhibitor –140. AltHowever, only studies with limited patients were eligible, therefore future research should focus on these less invasive methods.In the 100 most recent patients in our center, pre-transplantation nephrectomy was performed in 21%. Common indications were steroid-resistant nephrotic syndrome (38%) and large polycystic kidney volume (14%).We conducted a successful medical nephrectomy using ACE inhibitor in 6 out of 8 patients with nephrotic syndrome .Urological complications after kidney transplantation can be divided in early (4%) and late complications (9%). Urinary leakage (2%) and early ureteral stenosis (1%)  or lymphocele needing drainage occur in the 1st month after transplantations, whereas late complications are mostly UTI (15–58%) and late ureteral obstruction (5–8%) , 141–145The placement of a temporary ureteral stent remains controversial. Previous literature showed that stenting was associated with increased risk on BK viremia and UTIs , 148. ThIn adults, JJ stenting was shown to be preferable to percutaneous stents in terms of recovery, however duration of drainage remains debatable .A Cochrane Review in the adult population showed that early removal (<15 days) of bladder indwelling and per-urethral stents might decrease the risk on UTI, however differences are small . The benAdditional clinical trials are needed to support this practice.Vascular complications are an important cause for early graft loss and include mainly renal thrombosis (3–12%) and arterial stenosis (3–15%) , 155.Venous thrombosis is considered the most common cause of early graft loss and especially small children are at risk for developing thrombotic complications , 156. HoRenal artery stenosis is associated with hypertension and progressive graft dysfunction and can be due to kinking or trauma of the artery, vascular rejection, inadequate suturing or atherosclerosis.The most important lymphatic complication after transplantation is lymphocele, which occurs in 0.5–22% of the recipients , 160–162Lymphocele can result in compression of the graft vessels, ureter or bladder outlet and therefore cause decreased graft function. Analysis of the aspired fluid can differentiate from hematoma, urinoma and seroma. In pediatric recipients, a higher age, BMI and number of transplantations were associated with the development of lymphoceles . In addiAccording to our protocol, all children have a ureteral splint for 5 days in patients older than 4 years and 7 days in patients <4 years and a transurethral or suprapubic catheter for 7 and 9 days, respectively. Protocol antithrombotic prophylaxis for patients older than 12 years exists of daily 2500 IE dalteparine post-operative until good mobilization. Specific anticoagulants such as Direct Oral Anticoagulants (DOACs) or Vitamin K antagonists are given on indication. We don't use prophylaxis for arterial thrombosis. In total, 13 patients (3%) lost their graft due to thrombosis, all before 2005.The ureteroneocystostomy (UNC) technique is one of the surgical factors that might influence the urological complication rate .The method used for the neo-ureteral-bladder anastomosis has changed over the years in the adult population. Where an intra-vesical (anti-reflux) technique was common in the past, nowadays this has changed to an extra-vesical approach with or without anti-reflux technique .Overall, UNC techniques can be divided in being either intra-vesical or extra-vesical and refluxing or anti-refluxing .With the intra-vesical Leadbetter-Politano (PL) technique an anti-refluxing tunnel is created to prevent vesico-ureteral reflux (VUR) . This teAfter some time the easier extra-vesical modified Lich-Gregoir (LG) technique gained more popularity. In the LG technique, a single cystotomy is performed and the distal ureter anastomosis to the bladder is covered by detrusor muscle with the intention to create a valve effect and prevent VUR . Over tiCurrently, the “full thickness” method is commonly used in the adult population which has no anti-reflux mechanism . This teLittle is known about the optimal UNC technique in the pediatric population. Although an anti-reflux technique might be favorable regarding the increased risk of VUR in pediatric recipients , it coulThe importance of VUR as a complication after kidney transplantation is debatable since it is often asymptomatic and might not influence long-term outcome in adults , 175. ThAltogether, the choice of UNC technique in pediatric kidney recipients remains difficult and the long-term effects are still unknown. It would be worthwhile to compare outcomes of anti-reflux vs. reflux techniques in pediatric kidney recipients specifically.In our center, from 2000 all anastomoses were created using a refluxing extra-vesical technique. In 100 recipients that received their graft between 2002 and 2018 55% had least one UTI, 20% had recurrent UTI's. VCUG was done in 11 symptomatic children of which five were diagnosed with VUR.in situ or performing a graft nephrectomy (GN).As stated before, graft failure is mainly the result of rejection or thrombosis. When a graft fails, one has to choose between either leaving the graft It is well–established that GN is indicated in cases of vascular thrombosis, hyperacute rejection, and therapy resistant malignancy –187. RelIn adults, GN is performed in about 35% of patients with a failing graft , 190. InConsidered benefits of GN include reduction of inflammation, discontinuation of immunosuppression and possible reduction of the number of donor specific antibodies. However, surgery for graft removal may cause considerable peri- and post-operative morbidity such as inflammation and hemorrhage. Post-operative mortality rates ranged from 1 to 39% and was mostly caused by sepsis (17%). Moreover, re-transplantation outcomes are worse after GN compared to no GN , 194.Moreover, GN is associated with higher donor specific antibodies because of the potential absorptive capacity of the graft . PreviouTo establish the impact of GN on both allosensitization and graft outcome remains challenging because of multiple confounding factors. However, timing of GN is thought to be an important factor. Sener et al. showed that patients that had GN in the 1st month after transplantation had lower panel reactive antibodies (PRA) and reduced risk on future graft failure compared to those who did not have GN .In contrast, patients who had late GN (>1-year) were at increased risk for future graft failure and had increased PRA , 198. WaThe (dis)continuation of immunosuppressants after GN remains debatable, since continuation would prevent allosensitization while increasing the risk on infections, vascular disease and malignancy , 199. InThere is only limited research on GN in children. Whereas, high rates of morbidity and mortality were seen in adults, outcome in pediatric GN was shown to be good. No major complications, re-operations and blood transfusions were reported in the few studies on pediatric recipients , 191.Renal artery embolization (RAE) was thought to be a minimal invasive alternative to GN as it results in less surgical complications. However, the risk on necrotic pyelonephritis and post-embolization syndrome are increased after RAE and RAE as monotherapy is not widely used . HoweverBetween 1977 and 2020, 379 transplantations were performed in our center. Graft failure occurred in 108 grafts so far of which 66 (53%) were removed. There was no operative mortality and 32% of the surgeries resulted in complications which were all resolved .Subclinical acute rejection was shown to be a cause for deterioration of graft function which implies that early diagnosis and treatment is favorable. Since subclinical rejection can only be diagnosed by means of graft biopsy, the practice of “protocol” or so called “surveillance” graft biopsies is under debate , 205. AlLiterature on protocol biopsies in pediatric kidney recipients is scarce and future (randomized controlled) trials or small group trials are needed to address significance of early subclinical rejection and therapeutic interventions.Besides, possible benefits of protocol biopsies should be weighed against the potential risks such as arteriovenous fistulas and bleeding , 208. BeAlthough new randomized controlled trials or dedicated small group trials could provide valuable insights in this debate, future research should also focus on developing non-invasive methods for detection of subclinical rejection.Acute rejection of the kidney graft can be divided in either anti-body mediated rejection and T-cell mediated rejection . TherefoThere are several strategies in the treatment of T-cell mediated rejection. Traditionally intravenous pulses of methylprednisolone are used. Other options are polyclonal antibodies such as ATG, monoclonal antibodies against lymphocyte receptors such as alemtuzumab and rituximab and a proteasome inhibitor such as Bortezomib .Nowadays, the immediate use of polyclonal antibodies instead of methylprednisone is debatable. There is currently little evidence favoring one specific strategy and clinical decision making remains challenging.There are various possible strategies to treat anti-body mediated rejection, however the optimal therapy remains controversial. Some of the treatment options are similar as in T-cell mediated rejection such as polyclonal antibodies and methylprednisolone. Other possibilities are plasma-exchange, administration of intravenous immunoglobulins (IVIG) and the monoclonal antibody rituximab .In the past, plasmapheresis was common practice whereas this has a less prominent place in anti-rejection therapy nowadays. Billing at al. introduced the combination of IVIG and rituximab which reduced donor specific antibodies and stabilized renal function . AlthougApart from anti-rejection therapy, changes in maintenance therapy should be considered .In our center, graft biopsies are performed on clinical indications such as a deterioration of GFR. Five percent of our latest 100 pediatric kidney recipients had a graft biopsy during their transplantation admission. During a median follow-up of 47 months, 42 patients had at least one graft biopsy that mostly revealed calcineurin toxicity (32%) and acute rejection (30%). In total, 36 patients received pulsatile methyl prednisone during follow-up. We have reported one case of life-threatening respiratory failure after alemtuzumab administration .One of the factors that improved graft survival is the substantial change in immunosuppressive strategies over time.In the early days of pediatric kidney transplantation, immunosuppression consisted of total body irradiation and splenectomy which resulted in infection related mortality up to 72% , 4. Thisazathioprine (antimetabolites). After the introduction of calcineurin inhibitor (CNI) cyclosporine in the 1980's, graft survival increased substantially , an inhibitor of inosine-5′-monophosphate dehydrogenase) were introduced and the twenty-first century welcomed mammalian target of rapamycin (mTOR) inhibitors like sirolimus and everolimus  , 226. ThAlthough corticosteroid withdrawal seems safe in a selected population, long-term effects should be studied before general implementation.Basiliximab (a chimeric (human/murine) monoclonal antibody) or Alemtuzumab   .In our center, the use of cyclosporine and prednisone decreased over time, which is comparable to international literature . The Trap < 0.01), more CMV infections (11 vs. 0% p = 0.03) and more rejections (18 vs. 0% p = 0.02) than those on a steroid-sparing regimen. Other side effects did not differ between steroid-sparing and steroid-containing regimens , Epstein-Barr virus (EBV) and BK-virus (BKV). The latter specifically harms the graft.Without prophylaxis, these infections are most likely to occur in the early months after transplantation because of transmission trough the graft and high dosages immunosuppression during this period , 229.The use of CMV prophylaxis is recommended in several guidelines, however which patients should receive prophylaxis and for how long remains debatable , 230. VaIn our center, high risk patients (D+R-) receive CMV prophylaxis until 3 months after transplantation. In the 100 most recent transplanted patients, 62% used CMV prophylaxis. During a median follow-up of 47 months 14% developed CMV disease regardless of prophylaxis.Medication adherence is commonly defined as when the patient follows recommendations and instructions from health care professionals concerning the taking of medication that were previously agreed on .Medication non-adherence (MNA) to immunosuppressant regimens is an important factor limiting graft survival. Moreover, in adolescents (aged 11–21 years) it is considered the most important modifiable risk factor for graft loss , 233. ThThe overall prevalence rate of MNA is considered between 30 and 50%, however numbers vary according to the characteristics of the patient population, definition of MNA used, timing of measurements and methods used to assess adherence , 236. DePrevious studies reported a wide range of assessment methods like pill counts, questionnaires, patients' diaries, and random measurements of blood drug concentrations. Despite the several methods available, it remains difficult to assess MNA because all methods have their own limitations . In geneDespite its limitations, electronic monitoring is currently accepted as the most reliable measurement of adherence . In adulThe WHO has classified the factors of MNA in 4 categories: individual level, family level, health-care system, and community level . Most stAt the individual level, recipient age was found to be one of the strongest factors affecting the risk for MNA , 243. QuTransition of responsibility remains a difficult topic in this age category as these teenagers often desire more independence . In a DuAlthough many adherence-promoting methods were developed, single strategies were not shown to be effective , 247. PrAlthough some trials with these multi-component interventions have been conducted successfully, these were labor-intensive and not easy to incorporate in daily life , 249.Studies with electronic pillboxes and eHealth interventions have shown promising results in studies among patients with chronic diseases. However, these should be tailor made for this specific population , 250–252In addition, special attention should be drawn to the transition of recipients with delayed development. Since they will need extra support, they are prone to fall between two specialties due to their individual transition requirements, especially if they have comorbidities. Taken together, these results suggest that simple “one size fits all” interventions are not effective and that future interventions should be multidimensional and targeting risk levels on various levels in the healthcare system.In our center, medication adherence is addressed every outpatient visit, especially in adolescents. To support transition to adult care, patients are actively prepared from the age of 12 years in a personalized manner. Aspects of this transition phase are education on their disease, the different drugs, outpatient clinic visits with the first part of the visit without their parents. These patients are guided to take responsibility for their treatment. However, actual data on adherence are missing. Little is known on the non-adherence rates in our center.In the past decades, innovations in pediatric kidney transplantation led to increased graft and patient survival. Despite these innovations, we endeavor to optimize clinical care for pediatric kidney recipients. In this article we provided a state of the art overview and identified paucity of evidence on several important issues in comparison with our results. Future possibilities to improve pre-, peri-, and post-operative care are discussed below.Choosing between an old well-matched living donor and young poorly-matched deceased donor requires consideration of multiple aspects such as sensitization, existing waiting list time and the risk of graft failure . Future In order to fill in the knowledge gaps mentioned above, thorough and well-designed studies are needed. Previous literature is mainly based on retrospective studies in relatively small cohorts. Since the incidence of graft loss has impressively decreased, large volumes are needed to draw conclusions on the factors leading to graft loss.Besides, research in such a rapid changing field is challenging since data are quickly outdated and confounding factors such as innovations in practice are difficult to correct for.A possible solution for these problems might be collaboration between the different (inter)national registries. Currently, multiple organizations and registries are actively studying pediatric kidney transplantation such as the NOTR  and CERTAIN registry . However, each registry collects different data in a slightly different population. Collaboration between those registries allows studying large volumes of data and comparison of practices among different countries. A promising phenomenon is the development of European Reference Networks (ERN), which allows cooperation at the European level between clinicians with specialized expertise. They aim to improve diagnoses and treatment for patients with rare diseases and/or complex conditions. The ERN eUROGEN aims to improve diagnosis, create more equitable access to high-quality treatment and care for patients with rare uro-recto-genital diseases and complex conditions needing highly specialized surgery . ERKNet Surgical techniques have improved over time which resulted a reduction of peri-operative complications. Transplantation in small children is possible although blood pressure and perfusion are vulnerable. Peri-operative monitoring enables strict regulation of those parameters and a multidisciplinary team working according well-defined protocols is mandatory , 14, 76.Although in adults, a refluxing ureteroneocystostomy was shown to be comparable to anti-refluxing methods, less is known about the pediatric population. To reveal this topic future multicentered research should focus on differences in long-term outcome regarding graft function, UTIs and urological interventions. Thresholds for such studies are the confounding factors that differ between centers and the lack of routine VCUG to determine the rate of (asymptomatic) VUR.Immunosuppressive protocols have dramatically changed over time. The withdrawal of steroids was shown to be safe in low-risk patients regarding both graft survival and side effects. This remains uncertain for high-risk patients and long term outcome needs to be established.It remains challenging to find the best combination of immunosuppressive agents, as the balance between preventing rejection while limiting side effects is precarious. Future research should focus on the long term effects of immunosuppressive medication, especially regarding long term side effects. Biotechnical advancements might result in withdrawal of conventional immunosuppression. Current studies focus on cell-based therapy, which aims at the induction of donor-specific unresponsiveness in the setting of either operational tolerance or mixed chimerism .Medication non-adherence has been increasingly recognized as a cause of graft failure, especially in children and adolescents , 231. A Nowadays, artificial intelligence (AI) plays a major role in daily life. Application of AI in medicine and research is becoming more common, especially regarding risk assessment. Machine learning techniques are shown to be promising in processing biomedical data where they are successful for predictive models, image processing and genomic data analysis .In adults, AI has been successfully implemented in the field of kidney diseases. Kuo et al. . designeHowever, such models should be based on a representative cohort of patients, which is a problem in pediatric kidney transplantation. Because of the small patient volumes, development of reliable algorithms and proper validation remains challenging. Although many countries do have their own databases, international collaboration is needed as well as standardization of data, identification of the patient and linking between the different registries .Whereas, optimization of current care remains of utmost importance, recent technologies might offer new perspectives to renal replacement therapy. Current research focusses on developing a wearable artificial kidney that would improve both quality of life and quality of dialysis , 261.de novo has been studied over the last decades (Another promising field is that of regenerative medicine, the process of generating a human kidney  decades –265. Sev decades , 267. Di decades , 268.Despite these promising developments, there are many hurdles to take before one could generate a functioning human kidney. Among them are the possible tumorgenicity, the small scale of organoids and absence of potent vascularization and urinary drainage system , 270. ThAnother solution for the graft shortage might be xenotransplantation with the kidney of genetically modified pigs. Recently, surgeons have placed such a kidney in a brain-dead patient for research sake . Both thThis overview of 50 years care for pediatric kidney recipients revealed an impressive improvement of graft and patient survival. Important developments are the increased use of living donors, improved immunosuppressive therapy and better peri-operative care.Still, many questions remain unanswered. In our center, pre-transplant treatment modality, donor age and HLA mismatching did not affect graft survival which might advocate donor pool expansion. More large scale, multicenter studies are needed to confirm these findings.Since urological complications are more common in children, an active screening program for LUTD should be considered. Moreover, the optimal method for surgical vesico-ureteral anastomosis still needs to be established.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.LO: collected the data, performed the analysis, and wrote the paper. CB-R and LW: conceived and designed analysis, collected the data, and provided intellectual guidance. EC: collected the data and provided intellectual guidance. WdF: conceptualization, writing—review and editing, and 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.


Sorghum has good adaptation to drought tolerance and can be successfully cultivated on marginal lands with low input cost. Starch is used in many foods and nonfood industrial applications and as a renewable energy resource. Sorghum starches with different amylose contents affect the different physicochemical properties. In this study, we isolated starches from six sorghum varieties  and investigated them in terms of their chemical compositions and physicochemical properties. All the starch granules had regular polygonal round shapes and showed the characteristic “Maltese cross”. These six sorghum starches showed an A-type diffraction pattern. The highest amylose content of starch in Jinza 127 was 26.90%. Jiaxian had a higher water solubility at 30, 70, and 90 °C. From the flow cytometry analysis based on six sorghum starch granules, Liaoza 19 had a larger and more complex granules (particle percentage (P1) = 66.5%). The Jinza 34 starch had higher peak (4994.00 mPa∙s) and breakdown viscosity (4013.50 mPa∙s) and lower trough viscosity (973.50 mPa∙s). Jinnuo 3 had higher onset temperature, peak temperature, conclusion temperature, gelatinization enthalpy, and gelatinization range. The principal component analysis and hierarchical cluster analysis based on classification of different sorghum starches showed that Jiniang 2 and Jinnuo 3 had similar physicochemical properties and most divergent starches, respectively. Our result provides useful information not only on the use of sorghum starches in food and non-food industries but for the great potential of sorghum-based intercropping systems in maintaining agricultural sustainability. Sorghum bicolor (L.) Moench) has been widely grown in China for at least 40–50 centuries in arid, semiarid, and water-logged regions. Although a production zone is wide, the major growing areas are concentrated in the northern and northeastern parts of the country. Chinese sorghum belongs to a unique “kaoliang” group characterized by adaptation to the Chinese environments [Sorghum . The contents of protein, and starch in sorghum starches were 0.80–1.20% and 85.26–96.42%, respectively. Jinnuo 3 had the highest protein content compared with the percentages in other sorghum starches. The high starch content in these six starches showed that the Chinese sorghum varieties were ideal starch resources. The findings on fat and protein contents in sorghum starches were low, which indicated that the extracted starches were pure. The contents of amylose in Jinza 34, Liaoza 19, Jinnuo 3, Jiza 127, Jiniang 2, and Jiaxian were 17.61%, 22.12%, 8.60%, 26.90%, 8.30%, and 20.47%, respectively. These results indicate that the starches of Jinnuo 3 and Jiniang 2 had the lowest amylose content than the others.The chemical compositions of sorghum starches are shown in The morphology of different sorghum starches were observed via polarized light examination and scanning electron microscopy . PolarizFiji ImageJ program is useful as a practical and research tool in observing the physics structure of materials and scientific images ,25. The The flow cytometry is currently widely used as an effective method for the classification of starch granules. In this study, starches were isolated from six sorghum cultivars and initially classified into various subgroups by flow cytometry and then collected through flow sorting to observe their morphology by SEM  and partThe X-ray diffraction diagrams of the starches are shown in The light transmittance of starch, a one of the starch paste parameters, and the manufactured of starch-based products with high quality light transmittance are well known among popular amongst the consumers. p < 0.05) than that of the other sorghum varieties. The solubility of Liaoza 19 was relatively higher (p < 0.05) than that of the other sorghum varieties at 50 °C. p < 0.05) than that of the other varieties at 30 and 50 °C. The swelling power of Jiza 127 sorghum variety was higher (p < 0.05) than that of the others at 70 and 90 °C. A rapid rise in solubility (%) and swelling power (g/g) was found from 70 to 90 °C, at which gelatinization occurred. Lin et al. [The swelling power and water solubility parameters are shown in n et al.  reportedn et al.  reportedThe pasting properties of different sorghum starches are shown in The gelatinization transition temperature start (To) ranged from 68.04 to 76.11 °C; peak (Tp) ranged from 71.34 to 80.77 °C; completion (Tc) ranged from 79.14 to 88.51 °C; enthalpy for gelatinization (ΔH) ranged from 5.94 to 13.38 J/g; and gelatinization temperature ranged from 10.9 to 12.4 °C . The higp < 0.05).PCA and HCA were carried out in this study to better understand the characteristics of sorghum starches . PC1 andThe HCA was performed on the basis of physicochemical properties of starches to compare the relationship of different sorghum varieties C. ClusteA heat map was created to show further correlation coefficients among amylose contents, starch contents, relative crystallinity, pasting, and thermal properties of sorghum starches. In the present study, amylose content had a significant positive correlation with SB, FV, BD, and PV, whereas a negative correlation was noted among Tp, Tc, and To in the starch samples . SimilarSix varieties of sorghum, namely, Jinza 34, Liaoza 19, Jinnuo 3, Jiza 127, Jiniang 2, and Jiaxian, were grown in the Experimental Fields of the Northwest Agriculture and Forestry University, Yulin, Shaanxi , China in 2021. The same sowing and environmental growth conditions were used to all planting sorghum materials. The seeds were separated from the impurities and other inert-materials for further use.w/v) at 4 °C for 24 h. The soaked grains were washed, the shells were removed, and the grains were crushed into flour for 3 min with a grinder . The suspension was filtered through 100 and 200 mesh screens, and the residues on the screens were washed with water. Grinding and filtering were performed three times. After cleaning, the residues were discarded, and filtration was performed for 1 h. The filtrate was rotated at 4000 rpm. The gray protein-rich top layer was removed with a spatula for 10 min. The contaminants were easily separated by adding 0.3% NaOH and centrifuged at 4000 rpm for 10 min. The procedure was performed with a wash tank containing double-distilled water, and centrifugation was again performed at 4000× g for 10 min. Washing and centrifugation were repeated several times until the top starch layer was clear. The final starch was dried in a fume hood for 24 h at 40 °C, and a 100-mesh sieve was used to clean the lumps. The extracted starch was stored in an air-tight container for further characterization.Starch was extracted using the alkaline steeping method of Singh et al.  and Gao Fat was obtained through Soxhlet extraction, and the solvent was petroleum ether with a boiling point of 60–90 °C. The protein was obtained using Kjeldahl method, and the content of protein (%) was calculated from the nitrogen content (N × 6.25). The total starch contents were obtained via the anthrone spectrophotometer method and calculated by multiplying the glucose concentration with the conversion factors . The amyw/v) and 50% glycerol was observed under polarized light. An Olympus BX53 polarized light microscope  was used. The suspension was photographed with a CCD camera .A suspension of starch 10% . The granule size distribution and average diameter of starch granules was observed by using of Fiji ImageJ software .The complexity and sizes of the starch granules were determined via flow cytometry , according to the method of Zhang et al. .The structure of the crystal test of starch was measured with an X-ray diffractometer . The Cu Ka X-ray wavelength was operated at an accelerating voltage of 40 kV and a current of 100 mA. The samples were scanned over the range of 5°–50° 2θ with a step size of 0.02° . The rel2O were mixed in a centrifuge tube and then placed in a 100 °C boiling water for 30 min. Then the mixture was centrifuged again under 5000 r/min for 15 min and the supernatant was discarded. The water holding capacity (WHC) of the starch granules was calculated as described by Yang et al. [According to the method described by Yang et al. , the ligg et al.  using thThe swelling power and solubility in water were measured using the method of Uarrota et al. . A 500 mStarch thermal property was measured by DSC . Approximately 3.00 mg of the sample was placed in an aluminum crucible with 2:1 deionized water and sealed with a matching base and allowed to stand for 2 h at room temperature. The sample aluminum crucible was heated in 20 °C to 110 °C at a rate of 10 °C/min. The sealed blank aluminum crucible was used as the control. Parameters recorded were gelatinization onset, peak, conclusion, temperatures and enthalpy, and gelatinization range.The pasting properties of starches measured with a rapid viscosity analyzer . The 3 g of dried starch was mixed with 25 mL of water and heated at 50 °C for 1 min and heated at a rate of 12 °C/min to 95 °C. The sample was kept at 90 °C for 2 min, cooled at a rate of 12 °C/min, and reserved at 50 °C for 1 min . Peak vip-value of 0.05 was considered statistically significant. The PCA was performed on the basis of the correlation matrix. The HCA was used in determining the link among the groups, and the Ward’s cluster method and Euclidean distance as the interval measures were used. Statistical software XLSTAT new version 2020.5 was used in determining the physicochemical properties of the sorghum starches.For sample characterization, measurements were performed at least twice unless otherwise specified. All data are represented as the mean values ± standard deviations. Data were subjected to one-way variance and Tukey’s multiple-comparison analysis by using IBM SPSS version 20.0 . A We studied the chemical compositions and physicochemical properties of starches from six sorghum varieties. All sorghum starch granules showed regular polygonal round shapes and exhibited typical “Maltese crosses”; however, they had different complex granules. The starches of Jiniang 2 and Jinnuo 3 had the lowest amylose contents than those of the other starches. These six sorghum starches all exhibited the same A-type diffraction pattern; however, the relative crystallinity of Jinza 34 was higher than the other starches. The six starch pastes had differences in water solubility, swelling power, light transmittance, and pasting and thermal properties. Jinnuo 3 and Jiniang 2 sorghum cultivars required more energy for starch gelatinization. Sorghum starch had good stability and is suitable for use as a frozen food thickener or food additive, and as a raw material for porridge, couscous, and mayonnaise. Under marginal lands with low input conditions, successful cultivation of crops is a common challenge and sorghum-based intercropping is one of the promising options for agricultural sustainability and rural livelihood security of small scale farmers in arid, semi-arid, and water-logged regions.