Improvement of energy harvesting performance from flexible thin film-based energy harvesters is essential to accomplish future self-powered electronics and sensor systems. In particular, the integration of harvesting signals should be established as a single device configuration without complicated device connections or expensive methodologies. In this research, we study the dual-film structures of the flexible PZT film energy harvester experimentally and theoretically to propose an effective principle for integrating energy harvesting signals. Laser lift-off (LLO) processes are used for fabrication because this is known as the most efficient technology for flexible high-performance energy harvesters. We develop two different device structures using the multistep LLO: a stacked structure and a double-faced (bimorph) structure. Although both structures are well demonstrated without serious material degradation, the stacked structure is not efficient for energy harvesting due to the ineffectively applied strain to the piezoelectric film in bending. This phenomenon stems from differences in position of mechanical neutral planes, which is investigated by finite element analysis and calculation. Finally, effectively integrated performance is achieved by a bimorph dual-film-structured flexible energy harvester. Our study will foster the development of various structures in flexible energy harvesters towards self-powered sensor applications with high efficiency. In recent years, energy harvesting technologies have drawn attention from many researchers hoping to establish self-powered sensors and Internet of Things (IoT) systems for future applications ,2. AmongAlthough some principles have been developed to convert mechanical energy to electrical energy, piezoelectric materials and devices have still been considered as prospective energy harvesting technology owing to the simple device structures and environmental robustness regardless of wear, humidity, and serious heaviness ,7. In adIn fact, triboelectric energy harvesting, which is based on the coupling between tribelectrification and electrostatic induction, is also a promising technology for self-powered sensor devices. Triboelectric energy harvesters can generate very high voltage signals ,16,17. IVarious technologies have been reported to fabricate piezoelectric ceramics-based flexible energy harvesters, such as the stamping transfer method, polymer-hybrid composites, single-crystalline ceramic lamination, and so on ,23,24,253, etc.), while the processing yield becomes low. Therefore, the original LLO process for the flexible thin film energy harvesters should be revisited to improve the device performance and stability. Similar to the technology of multilayer ceramic capacitor (MLCC), flexible thin film energy harvesters can also be enhanced by using layered piezoelectric ceramic films on a single area of plastic substrate. However, there has not yet been a systematic investigation into layered flexible piezoelectric thin film energy harvesters for effectively enhanced and integrated energy harvesting performance.Even though the performance enhancement of the LLO-based flexible thin film energy harvesters has been studied using the quantitative simulations and the textured polycrystalline piezoelectric films resulting from modifying basal mother wafers , the cryIn this study, we demonstrate two different types of flexible thin film energy harvesters as prototypes of multilayered  piezoelectric generators fabricated by the reported stable LLO process. One type is a stacked dual-film structure, while the other is a double-faced (bimorph) dual-film structure. Because both devices are fabricated by the guaranteed LLO process and piezoelectric ceramics, the intrinsic material properties are very reasonable and reliable. Nevertheless, the performance of the two structures is highly different due to the differences in mechanical strain and generated piezopotential of piezoceramic films. The stacked dual-film energy harvester cannot generate effective energy harvesting performance, while the bimorph energy harvester presents good energy harvesting performance and reasonably integrated energy harvesting output for performance enhancement. The important discrepancy originates from the location of mechanical neutral plane for applied effective strain in piezoelectric active layers. Thus, it is noted that the device performance cannot simply be improved by increasing the number of piezoelectric active layers in a single flexible film energy harvester. Our investigation provides an appropriate design and perspective for multilayered piezoelectric film-based flexible energy harvesters towards performance-enhanced self-powered mechanical sensor systems.0.52Ti0.48O3 (PZT) for the piezoelectric ceramics of the flexible film energy harvester because it is commonly used for various piezoelectric applications. In particular, the 52/48 composition is well known as the most effective piezoelectric composition of the PZT material system, called the morphotropic phase boundary (MPB) [We used PbZrry (MPB) ,33,34. TIt should be noted that PZT is the most widely used piezoelectric material for various applications because it has highly piezoelectric properties and relatively easy processes. However, it contains lead (Pb), which is very harmful to biological systems. Because of the risk of lead-based poisoning, a variety of lead-free piezoelectric materials have been developed, especially for biomedical device applications ,36.33 piezoelectric mode when the device is bent [31 mode in bending. For the PZT system, d33 is higher than d31. Note that the active area of a PZT thin film with electrodes is about 1.2 cm × 1.2 cm. It should be mentioned that the simple geometry or area of IDEs can be modified in comparison with previous reports [−1 through the IDEs.The fabrication steps of the stacked device are depicted as cross-sectional schematics in  is bent . The dim reports ,28,29,33−1 through the IDEs, but each PZT film should be poled by opposite polarity because the stress applied to each film must be opposite when the device is bent in measurement . Both devices were measured by a Keithley 6514 electrometer  and a customized bending machine stage based on a linear motor , as shown in The fabrication steps of the bimorph device are also described as cross-sectional schematics in For the stacked device, failure  easily occurs when the second PZT film is detached from the sapphire wafer after LLO because it should be aided by mechanical force applied by tweezers, which can induce the fracture of the underlying first PZT film. This problem can be solved by a method in which the second PZT film is transferred onto the first layer device so as not to overlap completely, as shown in To investigate the material characteristics of PZT films in detail, we compared the X-ray diffraction (XRD) patterns and Raman spectra of the as-deposited PZT film on the sapphire wafer, the first transferred PZT film, and the second transferred PZT films in the two different dual-film structures. + laser source as an excitation source at ambient temperature. The bands of the Raman shifts around 205, 275, 330, and 591 cm−1 correspond to the typical property of perovskite MPB phase of PZT [Raman spectroscopy also shows the well-retained MPB phase structure of PZT films during the multistep LLO processes, as presented by e of PZT ,33. The e of PZT . Note the of PZT . The RamA stacked structure is the first way to enhance and integrate the energy harvesting performance of PZT thin-film energy harvesters in a single device because various integrated devices exist in vertically stacked structures like the MLCC. Therefore, our stacked dual-film-structured flexible film energy harvester was investigated first.One might expect that each PZT layer can generate high-performance energy harvesting signals and the performance can be merged for higher voltage and current signals. As shown in In contrast, the bimorph dual-film-structured energy harvester generates the expected and high-performance signals in the same bending condition ,27. As pTo investigate the difference between the performance of the stacked dual-film-structured flexible energy harvester and the bimorph dual-film-structured flexible device, we performed the finite element analysis (FEA) simulations using COMSOL Multiphysics computation program . All matN is the total number of layers, it is the thickness of the ith layer (from the top), and is iY ≡ absolute Young’s modulus and υi ≡ Poisson’s ratio of the ith layer) [YSU-8 = 4.02 GPa, υSU-8 = 0.22, and tSU-8 = 5 μm; (2) YPZT = 62.5 GPa, υPZT = 0.35, and tPZT = 2 μm; and (3) YPET = 3 GPa, υPET = 0.4, and tPET = 125 μm.This serious discrepancy should be analyzed by the mechanical neutrality in the flexible device configuration. The position of the mechanical neutral plane in a layered device structure can be described as below Equation (1):h layer) . For ther is the bending radius and δ is the distance from the mechanical neutral plane [The mechanical neutral plane is ~48 μm below the top surface. Therefore, the distance between the mechanical neutral plane and the middle of first PZT film is about 35 μm. The applied bending strain in the PZT layer is calculated by Equation (2):al plane . The calIn contrast, the generated piezopotential of each PZT layer of the bimorph structure is generally high (~160 V), as shown in The computational result can also be analyzed by Equations (1) and (2) for the mechanical neutral plane and the actually applied strain. According to Equation (1), the location of the mechanical neutral plane in a layered device structure is ~69.5 μm below the top surface. Hence, the distance between the mechanical neutral plane and the middle of each PZT film is about 63.5 μm because the bimorph dual-film-structured device has the symmetric cross-sectional configuration. This means that the applied bending strain to the PZT layers of bimorph device is about 0.394% in the same bending stimulation, slightly higher than that of the general one-layer PZT thin film energy harvester ,33.−2.As declared previously, the performance enhancement of energy harvesting by signal integration in a single device is very important without any complicated multiple device arrangements or complex material modifications, in order to facilitate more rapid technological growth for commercialization of the advantages of LLO technology-based flexible thin film energy harvesters with high performance. Therefore, we integrated efficiently the generated signals from both PZT layers of the double-faced dual-film-structured flexible energy harvester for effectively enhanced performance. As presented by In summary, we have demonstrated two different dual-film-structured flexible thin film energy harvesters for effectively integrated energy harvesting performance: a stacked structure and a bimorph structure. The LLO process-based fabrication has been selected for the basic energy harvesting device because it was confirmed to realize flexible high-performance harvesting unit devices. However, there have still been only a few ways to enhance the performance and/or integrate the energy level of the flexible film energy harvesters, which is highly significant for the future progress of commercialization, sensor applications, and self-powered electronics based on piezoelectric device principles. Multiple device connection is not useful due to the complicated features. Material modification approach was too expensive to achieve the improvement of the flexible energy harvesting unit. Therefore, we have suggested integrated structures of PZT film devices in a single area, like the approaches of MLCC. Both stacked and bimorph structures of dual PZT thin films fabricated by multistep LLO processes are well established on the flexible PET sheet substrate. However, the stacked dual-film-structured flexible energy harvester cannot generate effective energy harvesting signals, while the bimorph structure shows the general performance of PZT thin film energy harvesters. Based on the theoretical simulations and calculations, it is well discussed that the phenomena is due to the ineffective position of mechanical neutral plane in the stacked dual-films structure. Therefore, we propose the bimorph structure to effectively integrate and improve the performance of flexible PZT thin-film energy harvester as a single device configuration. As we expected, the energy harvesting performance of the double-faced device are the voltage up to ~280 V and the current up to ~2.2 μA, which indicated the good performance enhancement and integration in the bending deformation. Because the piezoelectric energy device and technology can be directly associated with various sensor systems ,42,43,44


Comparison of CDDP-sensitive cases with CDDP-resistant cases, the expression of c-met, HMGB1, and PCDHB9 was correlated with CDDP resistance. Among them, the expression of HMGB1 showed the most significant correlation with CDDP resistance in multivariate analysis. Treatment of TMK-1 and MKN74 human gastric cancer cell lines with ethyl pyruvate (EP) or tanshinone IIA (TAN), which are reported to inhibit HMGB1 signaling, showed a 4–5-fold increase in inhibition by CDDP. Treatment with EP or TAN also suppressed the expression of TLR4 and MyD88 in the HMGB1 signal transduction pathway and suppressed the activity of NFκB in both cell lines. These results suggest that the expression of these cancer metastasis-related genes is also related to anticancer drug resistance and that suppression of HMGB1 may be particularly useful for CDDP sensitization.The role of metastasis-related genes in cisplatin (CDDP) chemoresistance in gastric cancer is poorly understood. Here, we examined the expression of four metastasis-related genes (namely,  The multidisciplinary treatment of gastric cancer has improved its prognosis. In this treatment modality, cis-dichlorodiammine platinum (CDDP) plays a central role along with 5-fluorouracil (5-FU) and taxanes in chemotherapy for advanced gastric cancer . ChemothGalluzzi et al. divided the mechanism of CDDP resistance into four types : (1) preCDDP is likely to be administered in patients with advanced gastric cancer, especially to those who have developed metastases. Some metastasis-related genes are highly expressed in primary tumors in gastric cancer with metastasis. The effect of these genes on CDDP sensitivity is unclear. Therefore, studies on the relationship between the expression of such genes and susceptibility to CDDP are necessary. c-Met gene amplification is frequently observed [c-Met gene locus correlates with peritoneal dissemination [PCDHB9 is a gene of the procadherin family, and its overexpression decreases cell adhesion and promotes peritoneal dissemination [c-Met is a receptor for hepatocyte growth factor and is frequently overexpressed in gastric cancer . Its expobserved . In addimination . HMGB1 imination , and itsmination . HMGB1 imination ,10. RegImination . RegIV pmination ,12. PCDHmination .c-Met, HMGB1, RegIV, and PCDHB9 with CDDP sensitivity, with an aim to reveal new ways to overcome CDDP resistance.Elucidating the involvement of metastasis-related genes in drug resistance is important in the treatment of advanced gastric cancer. In this study, we investigated the association of the expression of The expression of the four metastasis-related genes was examined by immunohistochemistry in 39 gastric cancer cases. In all cases, neoadjuvant chemotherapy was administered with CDDP alone or in combination as CDDP+5-FU. As shown in Upon immunostaining, positive staining was observed in the cytoplasmic membrane and cytoplasm for c-met, PCDHB9 and RegIV, and in the nucleus for HMGB1 . FurtherIn 39 gastric cancer cases, the protein expression levels of these genes were compared between sensitive and resistant cases . A signiHMGB1 showed the strongest correlation among the four genes. We also examined CDDP responsiveness based on the expression of the four genes and using the cut-off values in 10 biopsy specimens obtained before chemotherapy  were reported to induce HMGB1 expression in cancer cells and promote doxorubicin resistance . In addic-met gene expression by the anticancer drugs was different in the two gastric cancer cell lines, alteration of c-met expression is thought to depend on individual tumors. From these findings, it is considered that monitoring the expression of anticancer drug resistance-related genes after starting the administration of anticancer drugs is needed for predicting the acquisition of anticancer drug resistance. In the future, it is expected that the comprehensive reactivity of the expression of metastasis-related genes and the anticancer drug resistance will enable accurate prediction of reactivity to various drugs.Alterations in gene expression associated with anticancer drug treatment might lead to changes in anticancer drug sensitivity. In the four metastasis-related genes examined in this study, expression of RegIV and PCDHB9 was thought to be reduced by CDDP and 5-FU treatment, while c-met expression was found to be increased. This suggests that c-met might be involved in the acquisition of drug resistance associated with anticancer drug treatment. Since the upregulation of In summary, the expression of cancer metastasis-related genes is suggested to be associated with a decrease in CDDP sensitivity. Particularly, HMGB1 was strongly associated with CDDP resistance. Cancer metastasis and drug resistance are both barriers to cancer treatment and HMGB1 might be a promised molecular target for both in gastric cancer. Trials for HMGB1 targeted therapy should be proactive at the clinical level.A total of 39 cases of gastric cancer involving surgical resection with neoadjuvant chemotherapy at the Nara Medical University Hospital and histopathological review by the Department of Molecular Pathology, Nara Medical University, during 2001–2019 were analyzed in this study. As written informed consent was not obtained from patients for their participation in the present study, all identifying information was removed from patient samples prior to their analysis, to ensure strict privacy protection (unlinkable anonymization). All procedures were performed in accordance with the Ethical Guidelines for Human Genome/Gene Research enacted by the Japanese Government and with the approval of the Ethics Committee of Nara Medical University .A human gastric cancer cell line, TMK-1, was previously established from a fundic gland-type gastric cancer case . The welCell growth was assessed via a tetrazolium (MTT) dye assay, as previously described . ApoptosConsecutive 4-μm sections were immunohistochemically stained using the immunoperoxidase technique described previously , with prgapdh mRNA was also amplified for use as an internal control (NCBI Reference Sequence: BC025925.1).To assess human CLDN4 mRNA expression, RT-PCR was performed with following cDNA synthesis with 0.5 µg total RNA extracted using an RNeasy kit . The primer sets are listed in t-test by InStat software . Multiple regression analysis was performed using EZR program [p-value of < 0.05 was considered to indicate statistical significance.Statistical significance was calculated using a two-tailed Fisher’s exact test, a one-way ANOVA, and Student’s  program . A two-s


Periprosthetic joint infection (PJI) is a catastrophic complication of shoulder arthroplasty. Commonly used surgical treatments include one- or two-stage revision, but their effectiveness in controlling infection is uncertain. We aimed to compare re-infection (recurrent and new infections) rates; clinical measures of function and pain; and noninfection complication rates of one- and two-stage revision surgery for shoulder PJI using a systematic review and meta-analysis. We searched MEDLINE, Embase, Web of Science, and The Cochrane Library to February 2018. Longitudinal studies conducted in patients with shoulder PJI treated exclusively by one- or two-stage revision were eligible. No clinical trials were identified. Re-infection rates were meta-analysed using random-effect models after arcsine transformation. The re-infection rate (95% CI) in pooled analysis of eight one-stage studies (147 participants) was 5.3% (1.4–10.6). The corresponding rate for 27 two-stage studies (351 participants) was 11.5% (6.0–18.1). Postoperative clinical measures of function and pain were not significantly different between the two revision strategies. The pooled noninfection complication rate (95% CI) for one-stage and two-stage revision was 12.1% (6.1–19.5) and 18.9% (8.4–31.9) respectively. New evidence suggests one-stage revision is at least equally as effective as the two-stage in controlling infection, maintaining joint function, and improving complications in shoulder PJI. As with periprosthetic joint infection (PJI) of the hip and knee, deep PJI following primary anatomic total shoulder arthroplasty (TSA) is a catastrophic complication which affects between 0.4 to 2.9% of patients5. The incidence of infection in primary reverse total shoulder arthroplasty (RSA) is about six times higher compared with that of primary anatomic TSA6. Treatment of shoulder PJI is a challenging task with the key aims of infection control, pain relief, and restoration of joint function. There is considerable controversy as to the optimal treatment strategy to achieve these aims. Treatment options for PJIs following shoulder arthroplasty are similar to those for hip and knee PJI and they include: long-term suppressive antibiotic treatment without surgical intervention; debridement, treatment with antibiotics and retention of the prosthesis (DAIR); one-stage and two-stage revision; and resection arthroplasty. Choice of treatment strategy is largely based on the experiences of treating surgeons and data from hip and knee arthroplasty studies7. Though the treatment option depends on the chronicity of infection, organism isolated and its virulence, component stability and patient fitness for surgery8, the one- and two-stage revision strategies are preferred as they are associated with successful clearance of infection and good functional outcomes10. As with PJIs after hip and knee arthroplasty, the two-stage revision strategy is regarded as the standard treatment option for shoulder PJI because it is generally consistently associated with good rates of infection control12. However, this revision strategy may be associated with functional impairment; though some studies have reported improved shoulder function after two-stage revision12. The one-stage is emerging as a promising alternative option; however, it is not as popular as the two-stage strategy. With regards to infection control, the evidence has been inconsistent for one-stage revision. Whiles some series have reported good infection control13, a high risk of infection recurrence has also been reported14. Emerging data suggests that one- and two-stage revision strategies for hip and knee PJI are comparable in terms of infection control16. No randomised controlled trial (RCT) has compared the effectiveness of the one- and two-stage revision procedures for managing shoulder PJI. However, a number of studies have reviewed the existing evidence by comparing results of case series that have reported findings on any of these two revision strategies and the results have been mostly inconclusive. A qualitative and quantitative review of 30 studies that compared infection control and functional outcomes among treatment options for shoulder PJI concluded that the one-stage may be as effective as the two-stage revision strategy, though infection control was reported to be better with two-stage revision17. However, one-stage revision produced a higher mean Constant-Murley (CM) score compared with that of two-stage revision. In another review which was based on six one-stage and 13 two-stage studies, George and colleagues reported a higher infection eradication rate for one-stage revision compared with two-stage revision18. In a recent overview study of existing evidence, the authors concluded that two-stage revision is the recommended strategy for PJI of the shoulder, but that a one-stage strategy can be used if the infecting organism is identified pre-operatively3.For many people with severe gleno-humeral disease, fracture, avascular necrosis or rotator cuff wear, shoulder arthroplasty is considered the most effective surgical intervention for alleviating pain and disability. In the UK in 2016, about 7000 shoulder arthroplasties were performedThe current evidence suggests that the optimal treatment strategy for the management of PJI of the shoulder is uncertain. There were several features of these previous reviews which limited the generalisability of the findings. First, these reviews did not pool the evidence using appropriate meta-analytic methods, which should take into account the heterogeneity of the included studies. Second, because the reviews did not pool the evidence using standard techniques, potential sources of heterogeneity among the contributing studies were not explored and no subgroup analysis was conducted across relevant characteristics. Third, sources of biases such as heterogeneity and preferential publication bias were not assessed. Finally, several new individual studies have been published recently. In this context, using a systematic review and meta-analysis, we aimed to conduct a detailed and robust comparison of the effectiveness of the one- and two-stages revision strategies for shoulder PJI using re-infection as a primary outcome and under a range of study-level clinical characteristics. Secondary objectives included (i) comparing the effectiveness of the one- and two-stage revision strategies using other clinical outcomes such as measures of function, pain, and satisfaction as well as noninfection-related complication rates and (ii) to explore for potential sources of heterogeneity between studies and assess publication bias.20 (Appendix The review was registered in the PROSPERO prospective register of systematic reviews CRD42017082747) and it was conducted based on a predefined protocol and in accordance with PRISMA and MOOSE guidelines7082747 a21, American Shoulder and Elbow Surgeons (ASES) Shoulder Assessment score, Simple Shoulder Test (SST), University of California Los Angeles (UCLA) score (function component), Disabilities of the Arm Shoulder and Hand score (DASH), Penn Shoulder Score (function component), forward flexion, abduction, external rotation, and range of motion]; (ii) pain ; (iii) satisfaction [as measured by Penn Shoulder Score (satisfaction component)]; and noninfection-related complications .We included studies that reported recruiting patients with PJI following shoulder arthroplasty (anatomic TSA and/or RSA) and who were treated exclusively by a one-stage or two-stage revision strategy, and followed post-operatively for re-infection (defined as recurrence of infection by the same organism(s) and/or re-infection with a new organism(s)) and/or other clinical outcomes such as (i) function [as measured by CM score 22 and has been described in previous reports16. This tool uses eight pre-defined domains namely: a clearly stated aim, inclusion of consecutive patients, prospective collection of data, endpoints appropriate to the aim of the study, unbiased assessment of the study endpoint, follow-up period appropriate to the aim of the study, loss to follow-up less than 5%, and prospective calculation of the study size. For each item, the instrument assigns a score of 0 for “not reported”, 1 for “reported but inadequate”, or 2 for “reported and adequate”. These are then summed up into a total score. The global ideal score is 16.After the initial screen of titles and abstracts by one reviewer (S.K.K.), we acquired potentially relevant articles for detailed full text evaluation. Two independent reviewers  assessed each article using the inclusion criteria and any disagreements regarding eligibility of an article was discussed, and consensus reached with a third author (A.D.B.). The data was independently extracted by one reviewer (S.K.K.) using a standardized data collection and quality assessments were also conducted. A second reviewer (V.W.) checked these data with that in original articles. We extracted data on year of publication, study design, country and geographical location (continent), mean/median baseline age, proportion of males, type of index arthroplasty, type of revision surgery, use and type of spacer, revision surgery characteristics, period of follow-up after revision surgery, number of re-infection outcomes and participants, and measures of pain, function, and satisfaction. For multiple publications involving the same cohort or series, the study with the most comprehensive information was used. We also corresponded with study investigators to provide missing information where relevant. We assessed the methodological quality of included studies based on the Methodological Index for Non-Randomised Studies (MINORS), a validated instrument which is designed for assessment of the quality of non-randomised studies in surgery23 because of the use of binary data with low rates. Details of the method have been reported in previous reports16. Summary rates of re-infection were pooled using random effects models to account for the effect of between-study heterogeneity24. Quantification of the extent of statistical heterogeneity across studies employed standard chi-square tests and the I2 statistic25. Potential sources of heterogeneity by study-level and clinically relevant characteristics were explored using stratified analysis and meta-regression26. Publication bias was assessed using Egger’s regression symmetry test27. Noninfection-related complication rates  with 95% CIs were also estimated. Other clinical measures of function and pain were compared between the two revision strategies using descriptive statistics (Wilcoxon rank-sum tests). We employed Stata version 14  for all statistical analyses.The rate of re-infection  with 95% confidence intervals (CIs) was used as the summary measure across studies. Rates were estimated using the Freeman-Tukey variance stabilising double arcsine transformationThe computer search and manual screening of reference lists of relevant studies identified 708 potentially relevant citations. After exclusions based on titles and abstracts, 55 articles remained for detailed evaluation. The remaining 30 articles were pertinent to the review question and were included in the pooled analysis Fig. . OverallCutibacterium acnes (C. acnes) was the most commonly causative organism for PJI of the shoulder in the majority of eligible studies. Studies were carried out in Europe , North America (United States of America), and Asia (South Korea). The methodological quality of included studies ranged from 8–12.Table p < 0.001) . There was no statistically significant evidence of publication bias using the Egger test (p = 0.66).Eight studies comprising of 147 participants evaluated the one-stage revision strategy and recorded 12 re-infections on follow-up (Tables p < 0.001) with a 95% prediction interval of 0.0 to 40.3% , which was not explained by any of the study-level characteristics assessed in subgroup analyses , when we restricted the analysis to studies of the highest quality (≥11). Among the higher quality studies, the pooled re-infection rate (95% CI) was 10.4 , which was similar to the main finding. There was no evidence of publication bias (Egger’s p = 0.71).Twenty-seven studies comprising of 351 participants with PJI of the shoulder reported 57 re-infections following two-stage surgical revision Tables . The poop-value for meta-regression = 0.41).In a subgroup analysis that compared re-infection rates between the two revision strategies, no significant difference was observed (p < 0.001) and 18.9%  respectively. In meta-regression analysis, no significant difference was observed .Fifteen studies (4 one-stage and 11 two-stage studies) reported on complications following revision surgery and these included dislocations; humeral or clavicular fracture; humeral or glenoid loosening; haematoma; postoperative instability; radial nerve entrapment and palsy; non-union; glenoid baseplate failure; glenosphere dissociation; and arthrofibrosis Table . The pooThere were no significant differences in measures of function and pain between both revision strategies Table . HoweverWe have compared the effectiveness of one- and two-stage revision strategies for PJI following shoulder arthroplasty using a systematic review and meta-analytic approach. Our primary outcome was re-infection (recurrent and new infections) with clinical measures of function and pain as well as noninfection complication rates employed as secondary outcomes. Pooled analysis showed that one-stage revision was associated with a markedly lower re-infection rate (5.3%) compared with the two-stage strategy (11.5%), though the difference was not statistically significant. Re-infection rates were generally similar across several study level clinically relevant characteristics for two-stage revision. The noninfection complication rate was also lower for one-stage revision compared with two-stage revision, though not significantly different. Though there were no significant differences in measures of function and pain between the two revision strategies, scores for CM, forward flexion, and abduction were better in one-stage revision patients; whereas scores for ASES, SST, external rotation, internal rotation, and Penn Shoulder were better in two-stage revision patients. However, given the limited data for these clinical outcomes, the findings should be interpreted with caution.18. Functional outcomes as measured by CM score was also better for the one-stage revision. In a systematic review including 15 studies, Marcheggiani and colleagues reported significantly better infection control rates for one-stage revision compared to two-stage revision, with no difference between CM scores28. Nelson and colleagues reported higher infection control for two-stage revision; however, in their systematic review including 30 studies, the difference between the two strategies was not significant17. The CM score for one-stage revision was also better but not significantly different. In our study, we employed a robust meta-analytical approach which took into account appropriate weighting of studies, whereas previous reviews simply summed the number of patients and number of infections, an approach which gives misleading results29. In addition to reporting data on noninfection complication rates and other measures of function as well as pain, we were careful not to include studies with overlapping participants; an approach that was ignored by the previous reviews. For example, the study of Klatte et al.30, included participants that had been included in the two studies of Ince et al.31. We also only included studies of patients with PJI following a shoulder arthroplasty as the index procedure and not other procedures such as rotator cuff surgery and internal fixation; as inclusion of these could have biased our results. In the absence of clinical trial data, our results provide up-to-date reliable evidence on the comparative effectiveness of the two revision strategies, as they are based on a larger number of studies and outcomes and employed a meta-analytic approach taking into account the heterogeneity between contributing studies.In this comprehensive quantitative review, we report several relevant findings that have not been previously reported. Some of our findings are also consistent with that of previous reviews on the topic. Consistent with our results, George and colleagues in their review of 20 studies reported that one-stage revision was associated with better infection control compared with two-stage revision and the difference was not significant32. Noninfection complications were also lower for one-stage revision. There are no clear management guidelines or consensus as to which revision strategy to use for PJI of the shoulder. Considerable research into the treatment of PJI has been carried out in patients with lower limb arthroplasty and this body of evidence has provided insight for treatment of shoulder infection7. However, infections in shoulder arthroplasty are characterised by different infective organisms, signs and symptoms, laboratory data, and also run a different clinical course33. The treatment of PJIs in the shoulder joint is indeed a challenging task for both the surgeon and the healthcare system. George et al. report that unlike PJI after hip and knee arthroplasty, the two-stage revision is not considered the “gold standard” for the management of PJI following shoulder arthroplasty, as there are other treatment options which report favourable outcomes18. However, the two-stage revision is generally considered as the standard treatment. The two-stage procedure which is considered the treatment of choice in patients who are medically stable35 and recommended when the infective microorganism is unknown3, has been commonly associated with successful eradication of infection in several case series37. However, this procedure frequently results in significant functional impairment38, given the requirement for two major surgical procedures. It is also costly for the healthcare system. The hospital cost of two-stage revision for the treatment of an infected shoulder arthroplasty is about two times higher than the cost of a primary shoulder arthroplasty39. The use of the one-stage revision strategy for managing PJI of the shoulder is becoming increasingly popular, though it has not gained the same level of momentum as with the treatment of PJI following hip arthroplasty. The main advantage of this procedure is that only one surgery is required, therefore it is associated with shorter hospital stay and generally shorter antibiotic duration32. The one-stage procedure is also associated with less tissue destruction and dissection, less patient anxiety, has the potential for better functional outcomes, and is associated with cost-benefits compared with the two-stage strategy40. Given the limited opportunities for further antibiotic therapy, the one-stage revision has commonly been adopted in select cases where the causative organisms have been identified pre-operatively41. Functional outcomes for one-stage revision have also been suggested to be dependent on the integrity of the rotator cuff muscles and type of prosthesis used41. Given the abundance of literature showing C. acnes as the common organism responsible for PJIs of the shoulder which is consistent with our findings, the potential to use the one-stage in a less selective manner is a possibility. Indeed, C. acnes is a gram-positive anaerobic bacillus which can be found in high concentrations in the acromion42 and can take up to four weeks to manifest as positive cultures43. PJI of the shoulder is associated with significant burden to the patient clinically and socioeconomically5 and also a challenge to the surgeon44. It also presents a huge financial burden to health systems39 and therefore a greater need to optimise resources. Indeed, previous reports suggest that shoulder PJIs have higher morbidity and costs compared with PJIs of other joints46. The current findings provide supportive evidence that the one-stage may be a potentially more attractive option for managing PJIs of the shoulders.Overall, findings from our study suggest that the one-stage revision strategy for the management of PJI of the shoulder is at least equally as effective as the two-stage in terms of infection control and maintenance of joint function. Indeed, the principal aim of treatment is to eradicate and prevent recurrence of infection as well as optimise joint functionIn addition to the several strengths enumerated above, our review employed a comprehensive search of several databases which yielded a large number of eligible studies compared to previous reviews on the topic. This ensured a more reliable comparison of the effectiveness of the two revision strategies in more detail than ever before. A detailed assessment of the methodological quality of the included studies was conducted using a well-established validated instrument. We adopted robust meta-analytic approaches which took into account the low event rates of the majority of the studies and heterogeneity between contributing studies. Other approaches included reporting of prediction intervals, comparison of re-infection rates among several clinically relevant characteristics, quantification of heterogeneity between studies and exploration of potential sources of bias. Our analyses showed no statistical evidence of publication bias and substantial heterogeneity between contributing studies for both revision strategies.47. Finally, there is a potential that data from national joint registries may be extremely useful in answering these questions, given their large-scale nature and cohort designs. However, no studies using registry data were eligible for the current review.Several limitations deserve consideration. There was a comparatively smaller number of one-stage studies and limited data on the secondary clinical outcomes, which precluded the ability to robustly compare the two revision strategies head-to-head. The sparse data also precluded detailed subgroup analyses by relevant characteristics which could have influenced the outcomes such as sex, indication for primary procedure, co-morbidities, type of infecting organism, type of implant, and antibiotic duration. We contacted several authors to provide additional data but received only one response. Given the nature of data reported by contributing studies, we were unable to distinguish between cases of anatomic TSA versus RSA, revision versus primary shoulder arthroplasty, as well as recurrent versus new infections. Given the limitations of aggregate published data, these findings should be interpreted with caution. However, the current findings are both timely and relevant and highlight the one-stage revision strategy as an equally or more effective option for treating PJI of the shoulder. In the absence of a carefully designed RCT powered for re-infection outcomes to robustly compare the effectiveness of the two revision strategies, access to individual level data from published studies may help to confirm the present findings. Our group has recently successfully employed this approach to compare the effectiveness of the one- and two-stage revision strategies for the management of PJI of the hipIn conclusion, new evidence suggests the one-stage revision strategy is at least equally as effective as the two-stage revision strategy in controlling infection and improving function and pain as well noninfection complication rates in PJI of the shoulder following joint arthroplasty.Supplementary Material


The major obstacle facing efficient gene therapy is the development of reliable delivery vehicles, which are both nontoxic and biocompatible and possess efficient cell-specific gene delivery. Previously, hybrid delivery vehicles comprising anionic liposomes and cationic polymers have been used successfully for gene therapy. In this study, hybrid vectors based on glycosylated artificial viral envelopes (including two novel compositions mimicking HIV and HSV envelopes) and polyethylenimine were morphologically and physiologically characterised. Transfection studies showed that the hybrid vectors based on the control liposomes, and their glycosylated modifications, had significantly higher transfection rates compared to the polyplexes. Improvement in the transfection efficiency was observed with the glycosylated HIV- and HSV-mimicking hybrid vectors, which also showed a safe biocompatibility profile based on the cytotoxicity and haemocompatibility assays. These glycosylated artificial viral envelope-based hybrid vectors could be used as safe gene delivery systems with potential to become new compositions for efficient nonviral gene therapy. Gene therapy is the therapeutic delivery of DNA, RNA, or antisense sequences into somatic tissue to prevent, modulate, or treat genetic and acquired diseases. This involves genetic diseases caused by a single mutation and acquired diseases for which no single gene mutation causes the disease state. The treatment is targeted at the cause of the disease rather than the symptoms. Designing an effective and safe delivery system, the deficiency of sustained expression, and an immune response are the major challenges involved in gene delivery. For therapeutic activity, a gene delivery vehicle should be biocompatible, protect its cargo from degradation, be nontoxic to the cells, be specific to the target tissue with minimal accumulation in the surrounding tissues and organs, and, most importantly, should not evoke immune responses. Viral vectors, such as retroviruses, adenoviruses, and adeno-associated viral vectors, have already been explored for their therapeutic potential in delivering genetic material. They have a more efficient gene transfer with long-term gene expression than most nonviral vectors, but some clinical trials have shown serious safety problems, such as acute immune responses, immunogenicity, and undesirable mutagenesis as well as restricted trans-gene size . NonviraLipids, polymers, and nanoparticles have been used in the recent past for the delivery of genetic material for therapeutic purposes ,4. A newIn this study, we formulated and investigated an advanced class of glycosylated lipid-polymer vectors, which mimicked the lipid composition of viral envelopes. The physiological composition of the artificial viral envelopes (AVE) made them serum-resistant and nontoxic. Due to their negative surface charge, different targeting ligands can be attached to the outer surface of the AVEs, such as viral-binding proteins and different sugar moieties . GlycosyPolyethylenimine (PEI), a cationic polymer known for its ability to efficiently transfect cells, has been employed as the polymer of choice in this study. While the branched variant of PEI is considered relatively cytotoxic, its linear counterpart is less cytotoxic due to the absence of primary amines . MoreoveThis aim of this study was to develop hybrid vectors based on glycosylated anionic liposomes that mimic natural viral envelopes ) for safe and efficient gene therapy. Different hybrid vectors, based on different AVEs, were developed using liposomes that were comprised of viral-mimicking lipids  and a 22 kDa linear variant of PEI. The hybrid vectors were characterised physicochemically and morphologically using dynamic light scattering, laser Doppler micro-electrophoresis, and atomic force microscopy, respectively. The effect of glycosylation with different glycolipid densities was evaluated as a function of transfection efficiency in vitro using a luciferase reporter assay. The toxicity and haemocompatibility of the hybrid vectors were investigated using MTT and clotting time haemolysis tests, respectively.2 at 37 °C and were passaged upon reaching 80% confluency.The lipids used for the formulation of liposomes were 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and 1,2-dioleoyl-sn-glycero-3-phosphoeth-anolamine (DOPE) and were a kind gift from Lipoid GmbH . The cholesterol was obtained from Sigma Aldrich . The glycosylated lipid DPPC-galactoside  was synthesised by U. Bakowsky , according to the method of Ogawa ,14. A fuLiposomes were prepared using the thin film hydration technique. For this, the lipids solutions (dissolved in a chloroform and methanol (2:1) mixture) were evaporated in a rotatory evaporator  to obtain a lipid cake. A total of 20 mM of HEPES buffer (pH 7.4) was added and the mixture was placed in an ultrasound bath above the Tm of the lipids to obtain nanoscaled liposomal vesicles. The liposomes were extruded  through 400 nm and 200 nm polycarbonate membrane filters  to obtain small unilamellar vesicles of uniform size. Polyplexes of PEI were formed by mixing appropriate amounts (N/P ratio of 10) of PEI and plasmid solutions and were incubated for no more than 30 min at room temperature before being used for further experiments. For lipopolyplex formation, the polyplexes were mixed with the liposomal formulations (polymer–lipid ratio of 2:5) by vigorous pipetting and were incubated for 1 h at room temperature. The formulations were characterised for their size (hydrodynamic diameter) and surface charge  using dynamic light scattering (DLS) and laser Doppler micro-electrophoresis techniques on a Zetasizer Nano ZS . Morphological analysis was performed by atomic force microscopy  using NSC16AlBS scanning probes  in the intermittent contact mode. The gene delivery efficiency of the formulations was evaluated using the luciferase reporter assay. Complexes containing 0.1 µg of pCMV-luc plasmid DNA were added to 96-well microtiter plates . Transfection studies were carried out in a serum-containing medium. After an incubation period of 48 h, the medium was removed and the cells were lysed using a cell culture lysis buffer . Ten mM of D-Luciferin  and luciferase assay reagent (Promega GmbH) were mixed together (1:20 ratio). This mixture was injected onto the microplate 50 µl/well) and the luminescence was recorded after 10 s on a plate reader equipped with an auto-injector . The corresponding protein content in the lysate was analysed using a Pierce BCA protein assay , in accordance with the manufacturer’s protocol. To determine the cellular uptake pathway of the hybrid vectors, pathway inhibitors inhibiting clathrin-mediated endocytosis  and caveolae-mediated endocytosis  were used [0 µl/wellToxicity studies were performed in a similar manner to the luciferase reporter assay with a slight difference that, after 48 h, the medium was removed and replaced with an MTT dye-containing medium. After 4 h incubation, the MTT dye was removed and the formazan crystals resulting from the metabolism of the MTT dye by viable cells were dissolved using DMSO. Absorbance was measured at 570 nm on a plate reader  with the absorbance of untreated cells (blanks) being considered as 100% viability.Haemocompatibility studies to determine the biocompatibility of the hybrid vectors for intravenous administration were performed using fresh blood from healthy donors drawn into citrate tubes, with their prior consent. The blood was centrifuged briefly to separate the plasma fraction from the erythrocytes which were subsequently washed with and diluted (1:50) with 0.9% NaCl solution . Complexp ≤ 0.05 were considered significant. Significance levels are denoted as * for p ≤ 0.05, ** for p ≤ 0.01, and *** for p ≤ 0.001.All experiments were performed in triplicate and the results are reported as averages of these measurements ± standard deviation. A two-tailed student’s t-test was performed to determine the significance of differences between the measurements. The probability values of With the aim of investigating the effect of glycosylation on gene transfer efficiency, liposomal formulations (both native and glycosylated) aimed at mimicking the HIV and HSV viral envelopes were prepared. Using a mixture of DOPE, DPPC (or DPPC-Gly), and DOPS, which are found in these viral envelopes ,17, lipoSince the artificial viral-like liposomes were ultimately meant to encapsulate the polyplexes, which were around 100 nm in size , extrusiAs the values of the physicochemical characterisation indicate, the hybrid vectors (formed using both native liposomes and their glycosylated counterparts) were the desirable size of <200 nm. The polydispersity index, in the case of the hybrid vectors, was slightly higher due to the presence of liposomes and polyplexes as a single entity. The net positive zeta potential could be attributed to the radiation of the positive charge of PEI to the outside of the liposomal bilayer, which is usually 4 nm in thickness [Atomic force microscopy studies were carried out, after letting the samples settle on a mica chip fixed on a glass slide. The sample excess was shaken off after a few minutes and the sample was allowed to air dry. The samples were visualised under the intermittent contact (tapping) mode to minimise any damage to the samples while acquiring an image. The scan rates of 0.5 to 1 Hz were used to acquire images of the polyplexes and hybrid vectors. The AFM micrographs  of the hA luciferase reporter gene assay was performed to assess the in vitro efficiency of the hybrid vectors. The hybrid vectors formulated using the PEI polyplexes were incubated with the cells for 48 h before being evaluated. The luciferase activity resulting from the transfection of the cells with the pCMV-luc plasmid is directly proportional to the transfection efficiency of the vectors. Compared to polyplexes, the hybrid vectors showed a tremendous increase in the transfection efficiency with more than a five-fold increase compared to gHV2 hybrid vectors A. As expToxicity studies were performed to determine the biocompatibility of the hybrid vectors. After the transfection of cells with polyplexes and hybrid vectors containing pCMV-luc plasmid DNA, the MTT dye was added to determine the percentage of viable cells that were capable of metabolising and converting the MTT dye into DMSO-soluble formazan crystals. The viability of the hybrid vectors was found to be above 85% C. LinearThe artificial virus-like nanoscaled hybrid delivery vectors designed using PEI polyplexes and viral-mimicking liposomes showed an improved transfection efficiency compared to PEI-based polyplexes. The glycosylated versions of these hybrid vectors showed further improvement in the transfection efficiency, which could be attributed to their dual internalisation pathways. These hybrid vectors showed a safe toxicity and haemocompatibility profile. This is a prerequisite for in vivo studies, which would be our prime research interest with the goal of making these hybrid vectors potential contenders for safe and efficient gene therapy.


Amphiphilic peptides can be self-assembled by establishing physical cross-links involving hydrogen bonds and electrostatic interactions with divalent ions. The derived hydrogels have promising properties due to their biocompatibility, reversibility, trigger capability, and tunability. Peptide hydrogels can mimic the extracellular matrix and favor the growth of hydroxyapatite (HAp) as well as its encapsulation. Newly designed materials offer great perspectives for applications in the regeneration of hard tissues such as bones, teeth, and cartilage. Furthermore, development of drug delivery systems based on HAp and peptide self-assembly is attracting attention. Peptide self-assembly allows new materials to be obtained through a bottom-up methodology . Within Multiple studies have been performed concerning the use of short peptide sequences as coatings, gels, and electroactive materials, as have recently been reviewed ,10,11,12Hydrogels are hydrophilic networks with a great capacity to retain water and have a great similarity with biological tissues. Hydrogels can be classified considering the type of cross-links  and their ability to incorporate chemical agents and cells. Furthermore, these systems can be designed to render shape memory effects, stimuli-responsive smart materials, and in situ gelling polymers. These are ideal for tissue regeneration since they can be used as injectable materials able to adapt to the form of tissue cavities. Applications of hydrogels have been extensively reviewed ,17,18, aHydroxyapatite (HAp) is a key component of hard tissues with recognized bioactivity, biocompatibility, and osteoconductivity. HAp has been extensively studied as an artificial bone substitute ,21, in aThe present review focuses on the recent advances in hydrogels based on peptides and utilized in biomedical applications related to HAp. To this end, we begin with describing the self-assembly of peptides and HAp characteristics in At present, self-assembled peptides are ideal systems for the development of new biomaterials. Various advantages that can be mentioned are as follows : (a) pepThe self-assembly process is mainly driven by thermodynamics, but kinetic factors also have a critical relevance concerning structural modulation and function integration, as reviewed by Wang et al. . The revα-Helices comprise both hydrophilic and hydrophobic amino acid residues, and are characterized by a secondary structure defined by a periodicity of 3.6 amino acids per turn. These helical motifs can be self-assembled into coiled-coil conformations due to their amphiphilic character . The maiCollagen-mimetic peptides have a great applied interest since collagen plays a fundamental structural role in both tissues and extracellular matrix (ECM) . Collageβ-Sheet peptides can be self-assembled into 3D-nanofibrous structures  when ionic self-complementary peptides are involved  34]. Ot. Ot34]. l,d-heterochiral peptides, (d) peptide amphiphiles (PAs), and (e) N-protected peptides.Self-assembled peptides able to form β-structures can be classified according to five main architectures: (a) ionic self-complementary peptides; (b) peptides having an alternate disposition of hydrophilic and hydrophobic residues ; (c) 3-CONH2 and negative (Ac-(LDLD)3CONH2 peptides. The derived β-sheets were thus constituted by double layers with hydrophobic and hydrophilic sides, the first ones being placed in inner pockets and the second ones exposed to aqueous environments. Ionic self-complementary peptides are based on the alternation of hydrophobic residues  and hydrophilic residues with positive  or negative  charges ,39. Thes2-CONH2 ..100].PA nanofibers containing units of serine or phosphoserine (PA-S) were able to nucleate carbonated HAp spheroidal crystals when exposed to a calcium-supplemented medium. These fibers could also be mixed (up to a 5 wt%) with peptide amphiphiles containing the biological adhesion epitope RGDS. The mineralized nanofibers were found to promote the osteogenic differentiation of human mesenchymal stem cells (hMSCs) .Anderson et al.  preparedMulticomponent hydrogels based on HAp and different ratios of two peptide building blocks, FmocFF and FmocR, were developed to obtain 3D scaffolds with tunable properties. These materials showed a high potential for bone tissue engineering due to their good biocompatibility and high cell adhesion .Different synthetic hydrogels have been developed for tissue applications, making it possible to achieve a great control of physical properties. Polymethacrylate and polyethylene glycol have been extensively considered as synthetic polymers ,104,105,Peptide hydrogels can achieve a high water content  and thixotropic properties ,107 sincGels based on PAs offer great advantages, such as the easy production of self-assembled three-dimensional nanofibrous networks. These appear optimum to favor cell diffusion, cell responsive degradation, and biological signaling . MechaniThe self-assembly of peptides can be initiated by screening their charged groups, which can typically take place through pH changes or the addition of multivalent ions to the physiological medium ,72. CyliFDFDFDFDFD-Pro is a clear example of an amphiphilic and anionic peptide that can form hydrogels by self-assembling into β-sheets. Its capacity to induce biomineralization is an added value for bone regeneration [FDFDFDFDFDGGGRGDS-Pro (FD-RGD)—a peptide that could be combined up to a 25 mol% with FD without disrupting the self-assembling fibril structure and increasing the density of osteoblasts cultured in the corresponding hydrogels [Pro-Dneration . This peydrogels . In factNote that small peptides can be incorporated into a polymer matrix in order to guide the biomineralization process. Genetically engineered peptides that selectively bind to inorganic compounds (GEPIs) have been developed by exposing a pool of random amino acid sequences (placed on the surface of a bacteriophage or bacteria host organism) to a target inorganic substrate ,122. The2 and β-glycerolphosphate (β-GP)  . The 20 Polymer–nanoparticle (PNP) materials constitute a new class of hydrogels that are formed through nonspecific interactions between polymer chains and the surfaces of colloidal particles ,128,129.Cationic nanoparticles can easily be prepared from amphiphilic peptides, and have been found highly effective as non-viral gene delivery vehicles to transport anionic nucleic acids into different cell types . NanoparSelected BPs have antitumor activity and inhibited tumor growth, migration, invasion, adhesion, and angiogenesis ,133. BPsThe maintenance of healthy bones, cartilage, and teeth is becoming one of the most relevant problems for an aging society, due also to intrinsic difficulties associated with the capability of old people to recover from fractures. Three components  must be considered in regenerative medicine ,135. ConHard tissue engineering has some inherent problems due to the limited number of cells that can be seeded effectively in the scaffold and also their uneven distribution that reduces the activity of incorporated cells in osteogenesis repair . ConsequBasically, new formulated bionanocomposite systems try to mimic the characteristics of the extracellular matrix: (a) a hybrid structure of inorganic matter  and organic macromolecules ; (b) a morphology based on particles with a high aspect ratio and diameter in the nanoscale range. Effective nutrient delivery is an important factor for tissue repair due to the strong metabolic demand of osteoblastic cells during tissue regeneration ,142. In w/v) and water that could have a similar behavior to the extracellular matrix. Specifically, Zhu et al. [Different porous scaffolds based on nHAp and chitosan (CTS) have been considered for tissue engineering applications. Nevertheless, some factors are not highly positive, the poor cell adhesion likely being the main problem limiting the material for an appropriate cell seeding. An interesting solution was the incorporation of a commercialized SAP hydrogel consisting of standard amino acids  contents were determined, with the detection of a clear growth of a mineralized extracellular matrix within the hydrogel. Mesenchymal stem cells were able to undergo a 3D differentiation to form mineralized matrices within the scaffold hydrogel .Several self-assembled RADA peptides  and RARADADARARADADA (modulus II) sequences, respectively) which easily form hydrogel scaffolds have been employed in tissue engineering ,146,147.−3), high porosity (5.17 m2 g−1) that allows vascularization and transport of nutrients, excellent biocompatibility and, consequently, high potential for bone tissue and biomedical applications.Li et al.  preparedN-(2-hydroxypropyl)methacrylamide (HPMA) copolymers grafted with two complementary β-sheet peptides  and Beta11B (Ac–TTEFTTTFETT–amide)) and a RGD motif  have also been designed for tissue regeneration [Complex systems  based onneration . Self-asneration . MoreoveIn vivo assays using different types of peptide amphiphiles were carried out by Mata et al. . A stronb-poly(propylene oxide)-b-poly(ethylene oxide)) and anionic and zwitterionic blocks) with attached HAp-nucleating peptides  at each end. These systems allowed a good control of mineralization and showed a liquid-to-solid transition at physiological temperatures. Thus, nanocomposites could be injectable, able to conform to the shape of bone or cartilage defects, and form solids at physiological conditions [Polymer–HAp nanocomposites were prepared using thermoreversible self-assembled polymer templates -nditions . Bone tissue engineering can overcome the problems inherent to the transplantation of allogenic bone grafts , pr8 and (VEVSVKVS)2 β-sheet-forming peptides [2 was able to retain the hydrogel structure at higher percentages of calcium ions  than (LE)8 due to its lower number of glutamic acid residues per molecule that prevents collapse. Viscoelasticity increased with calcium ion concentration, and it was possible to obtain an appropriate strength for a bone-filling material. Amorphous calcium phosphate (ACP) and HAp were mineralized along peptide nanofibers of (LE)8 and (VEVSVKVS)2 under neutral and basic pHs, respectively peptides . These wectively b,c.The enhancement of the scaffold bioactivity for bone regeneration can be achieved through the encapsulation of bioactive molecules . However, the release of these bio-factors is generally too fast to match the bone repair process, which requires several months to be completed ,160. TheEfforts have recently been focused on the development of peptides related to proteins having high osteoinductive properties, as it is the case of bone morphogenic protein-2 (BMP-2). In this way, problems related to its large size, insolubility, instability, high cost, and easy denaturation can be avoided. Thus, the bone morphogenetic protein-2 biomimetic peptide (BMPBP) has been proposed considering its reduced size  and the presence of typical SSPVT morphogenic sequences. Scaffolds encapsulating BMPBP were able to induce osteogenic differentiation and promote bone tissue regeneration ,164. HowGreat efforts are currently focused on the prevention of dental caries. These mainly involve actions to avoid the formation of microbial films and the enhancement of the effective remineralization process of the initial dental decay ,167. c-axis of HAp becomes aligned with the rod long axis). Ameloblast cells produce matrix proteins such as amelogenin, which self-assemble into nanospheres [Enamel is the hardest mineralized tissue of the human body and constitutes the external coating of teeth. This highly organized mineral is produced in the ectoderm germ layer and lacks collagen or other precursor proteins ,169. Theospheres  and reguospheres . Proteinospheres . The use of liquids and pastes that contain nHAp for the repair of tooth surfaces is highly extended for remineralization of submicrometer-sized enamel lesions . HoweverHuang et al.  develope2 sequence [+2 binding sites, which can act as nucleation points for HAp. After the application of P11-4 on the tooth surface, the peptide self-assembles due to the low pH in the lesion and provides a scaffold similar to the enamel matrix [Self-assembled peptides  have all matrix . 2, and finally coated with alginate to form three-dimensional scaffolds  and biodegradability properties. Scaffolds were found to induce osteoblast differentiation and to proliferate and attach to mouse preosteoblast MC3T3-E1 cells.Romanelli et al.  used Fmocaffolds . These nResin-based composites are usually employed for dental restoration, but the in vivo degradation of the adhesive–dentin interface is a recurrent problem that limits the lifetime of the clinical restoration . Viable Articular cartilage has a poor regenerative capacity, which is the main cause of the osteoarthritis disease typical of highly developed countries . This coCartilage-to-cartilage integration is more complex than typical bone-to-bone integration due to the presence of stem cells and vascularity. The mechanical performance of cartilage depends on some characteristics that contribute negatively to healing and integrating with tissues . Materia3-CNH2) hydrogel able to support the chondrogenesis of the encapsulated MSCs [Chondrocytes with a well-known low metabolic activity are main agents responsible for cartilage repair. The implantation of undifferentiated MSCs has a limited interest since, although a certain capability to promote the accumulation of cartilage-like tissue has been detected, the results are not sufficiently satisfactory to render a reliable regeneration of articular cartilage . The indted MSCs ,194 and A recent strategy consisted of the use of an osteochondral construct obtained from the interdigitation of HAp ceramic-based material with a functionally viable neocartilage prepared by self-assembly . GlycosaSuitable scaffolds for cartilage repair consist of three-dimensional templates in which attached or seeded chondrocytes produce and deposit a continuous ECM network. A great variety of natural and synthetic polymers are being considered for cartilage repair 3COQQRFEWEFEQQNH2 (P11-4) CH3COQQRFOWOFEQQNH2 (P11-8) and CH3COSSRFOWOFESSNH2 (P11-12) sequences have been evaluated. These SAPs can be mixed with chondroitin sulfate and initially delivered as a non-viscous fluid that can be triggered to self-assemble once located in place. Hydrogels showed promising properties for biomedical applications in glycosaminoglycan-depleted tissues. The best results were achieved with P11-8 and P11-8 samples.SAPs based on tryptophan and phenylalanine residues, which are placed on the middle and on the same side of the peptide to drive ribbon formation, and charged amino acids that favor and antiparallel sheet arrangement have been proposed . SpecifiThe number of works on the development of peptide-based hydrogels has increased exponentially since the discovery of the self-assembly capacity of molecules consisting of small peptide sequences. These hydrogelators have a wide range of applications, but the most promising ones concern the biomedical field, mainly as a consequence of the capacity of such materials to mimic the ECM. Properties like biocompatibility, establishment of physical and reversible cross-links, feasibility of cross-linking under physiological conditions, tunability, and trigger capacity justify the great interest in such materials. The research carried out so far highlights the diversity of appropriate sequences to induce self-assembly and the possibility of changing both structure and functionality. The great potential of such materials is obviously linked to the great variability in the composition and length of peptides, which opens the possibility of a tailored design to suit a specific property. The incorporation of charged amino acids in the peptide sequence promotes the interaction with calcium divalent ions, making it possible to use the derived hydrogels for the nucleation of HAp and even the development of nanocomposites. Promising results have been achieved in recent years concerning the development of hydrogels for the regeneration of hard tissues—mainly bone, teeth, and cartilage, as evidenced in the present review. The achievements attained in this field are very relevant, as it is expected that they will be able to reach an effective commercialization of these materials. The ability to gel under the action of suitable stimuli also allows the use of self-assembling peptides as injectable materials that can be fitted to the shape of the defect to be regenerated.Different challenges must still be overcome in order to obtain a maximum benefit and attain an effective commercialization. Progress is needed for the development of smart materials susceptible to external stimuli  and especially for drug delivery systems based on self-assembling peptides and HAp.


The significance of measurable residual disease (MRD) in hematopoietic stem cell transplantation (HSCT) is well recognized in different hematological malignancies, but the evidence indicate that pre-transplant MRD status is of particular importance in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). In ALL, inadequate response at the level of MRD is a commonly accepted risk factor for relapse and thus an indication for allogeneic HSCT. Similarly, growing evidence from the literature strongly suggest that MRD detected by multiparameter flow cytometry or molecular techniques should be also used for risk stratification in AML at the time of HSCT. Despite the well-defined association of MRD and outcomes of HSCT in acute leukemias, there are still many open issues such as the role of additional pre-transplant consolidation for MRD eradication, the ability of HSCT to overcome negative influence of MRD positivity on survival, the impact of conditioning regimen intensity on MRD clearance post HSCT, and transplantation outcomes or the selection of optimal donor with regards to MRD status. In addition, the role of MRD assessment in guiding post-transplant maintenance treatment should also be addressed in prospective trials. These open issues mostly awaiting further clinical studies will be discussed in our current review. In acute leukemias, measurable residual disease (MRD) is defined as the presence of residual leukemic cells in bone marrow (BM) or peripheral blood (PB) in patients who achieved morphologic complete remission (mCR). Notably, the new definition of LeukemiaNet for CR includes MRD negativity  indicatiThe significance of MRD in hematopoietic stem cell transplantation is well recognized in different hematological malignancies, but the evidence indicate that pre-transplant MRD status is of particular importance in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) as it correlates with the risk of relapse after hematopoietic stem cell transplantation (HSCT). The significance of MRD in acute leukemias has recently been highlighted in recommendations for diagnosis and management in ALL and AML published by different international groups of experts ,2,3,4.In ALL, inadequate response at the level of MRD is a commonly accepted risk factor for relapse and thus an indication for allo-hematopoietic stem cell transplantation  as was recently emphasized in the consensus paper by panel of international experts on behalf of the European Working Group for Adult Acute Lymphoblastic Leukemia (EWALL) and the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation .Similarly, growing evidence from the literature strongly suggests that MRD detected by MFC or molecular techniques should be also used for risk stratification in AML at the time of HSCT. The clinical significance of MRD status in AML has been confirmed recently by a position paper by the European LeukemiaNet (ELN) . Indeed,Despite the well-defined association of MRD and outcomes of HSCT in acute leukemias, there are still many open issues such as the role of additional pre-transplant consolidation for MRD eradication, the ability of HSCT to overcome positive MRD by achieving MRD negativity and the potential price in term of toxicity and transplant related mortality, the impact of conditioning regimen intensity on MRD clearance post HSCT, and transplantation outcomes or the selection of optimal donor with regards to MRD status. In addition, the role of MRD assessment in guiding post-transplant prophylactic and pre-emptive treatment should also be addressed in prospective trials. These open issues mostly awaiting further clinical studies will be discussed in our current review.2 is the most important adverse prognostic factor for treatment outcome, independent from “the old” classical risk factors identified at diagnosis [In acute lymphoblastic leukemia, detection of residual disease persisting below the mCR threshold of 10iagnosis ,6,7,8. Miagnosis ,6,7,8,9,iagnosis ,12,13.−4 to 10−5 [−5 to 10−6 [Sensitivities of clone-specific detection of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements using polymerase chain reaction (Ig/TCR PCR) range from 10 to 10−5 . However to 10−5 ,16,17. A to 10−5 . Yet, an to 10−5 ; therefo to 10−5 ,18. The  to 10−5 . With th to 10−5 ,21. Addi to 10−6 . Some au to 10−6 ,21,23. H to 10−6 . The fiv to 10−6 . The sumTCR and Ig gene rearrangements, it was demonstrated that the probability of disease-free survival (DFS) after 5 years was significantly higher for patients with persistent MRD > 104 who underwent HSCT in first CR than for those patients that did not undergo HSCT in first CR  [−3  assessed by RQ-PCR. In contrast, no benefit of HSCT on RFS was demonstrated in good MRD responders [MRD positivity in post-induction therapy is the universal strongest risk factor for relapse, regardless of the heterogeneity in MRD definitions applied, techniques used, and the time point being assessed in the different studies. Its negative impact on leukemia-free survival (LFS) may be at least partially overcome by HSCT, as strongly suggested by the results of all three cooperative trials conducted by the GMALL, PETHEMA, and GRALL research groups ,7,9. In Although outcomes of patients with persistent MRD who undergo HSCT is better compared with those who are treated with chemotherapy, the relapse rate after HSCT is significantly higher in MRD positive patients in comparison to those with undetectable MRD before transplant. Consequently, one can assume that eradication of MRD before HSCT may significantly improve the outcomes of transplant. A proof of principle is ALL. The efficacy of blinatumomab, a bispecific T cell–engager antibody in MRD eradication was evaluated in a single-arm study in adult patients with ALL in CR who exhibited MRD positivity after chemotherapy . A complThe other issue is the role of pre- and post-transplant MRD monitoring in guiding maintenance therapy after HSCT. This approach is intensively investigated in Ph-positive ALL. The use of tyrosine kinase inhibitors (TKIs) in post-transplant maintenance treatment results in reduced relapse incidence and improved long-term outcomes of HSCT, as was demonstrated by several prospective and retrospective studies ,29,30,31Furthermore, the development of highly sensitive techniques for MRD assessment may allow for revisiting the role of autologous transplantation in ALL. On behalf of the Acute Leukemia Working Party of EBMT, Giebel et al. retrospectively compared autologous versus allogeneic transplantation with myeloablative conditioning in patients with Ph+ ALL in first molecular remission and found no differences in outcomes . The autIn acute myeloid leukemia, genetic risk defined according to European LeukemiaNet (ELN) criteria is the most relevant factor in prognostication and decision-making processes ,34. HoweThe genetic and immunophenotypic diversity of AML is a major challenge for introducing an MRD assessment into daily clinical practice. In view of these difficulties, the ELN MRD Working Party has published guidelines for the use of MRD in clinical practice . The two−3 and 10−5 [−5 sensitivity can be reached mostly in experienced flow cytometry laboratories. MRD monitoring with MFC relies on defining leukemia-associated immunophenotypes (LAIPs) at diagnosis which is required in order to be able to detect MRD in the post-remission phase of the treatment. Alternatively, MRD assessment may be based on an analysis that screens for established aberrant differentiation/maturation immunophenotypic profiles at follow-up that is termed “different-from-normal” (DfN) approach [The most widely employed technique, applicable for more than 90% of the AML patients, remains the MFC with a sensitivity between 10and 10−5 ,38,39. Tapproach . The thiapproach ,41,42,43NPM1 mutation, RUNX1-RUNX1T1 and CBF-MYH11 in CBF-AML, and PML-RARA in acute promyelocytic leukemia [−4 to 10−6. However, these mutations accounts for no more than 50% of AML patients; in other words, RT-qPCR based MRD monitoring is not applicable for at least half of the AML patients [FLT3-ITD , FLT3-TKD , NRAS, KRAS, IDH1, IDH2, and MLL-PTD may be lost between diagnosis and relapse as a result of clonal evolution of leukaemia, thus being unstable throughout treatment. Therefore, ELN guidelines advise against using them as single markers, but they may be used to detect MRD in combination with other mutations [WT1) expression analysis in MRD assessment with the use of standardized and validated WT1 assay developed by ELN researches, who established reference ranges for WT1 expression in normal blood and bone marrow [WT1 encodes a transcription factor overexpressed in approximately 90% of AML patients, but the assessment of its expression for MRD monitoring has remained controversial due to limited data from large, controlled studies, as well as undetermined precise cut-off values and time-points for analysis. However, very recently researches from South Korea published a study on the optimal time-points and thresholds for the bone marrow WT1 expression assessment by real-time PCR with the use of the ELN normalized method in a large cohort of AML patients undergoing allo-HSCT [WT1 MRD assay useful for identifying patients with high risk of AML relapse.The main technical limitations of MRD monitoring by MFC result from (1) the hemodilution of the BM samples; (2) difficulties in proper gating of both pure WBC and blast cells, and (3) biological heterogeneity of the leukemic population. The 2017 ELN guidelines recommend RQ-PCR or NGS as alternative methods for MRD assessment. PCR-based methods  depend on identification of pretreatment-AML-associated mutation at diagnosis . RQ-PCR,leukemia ,36,45, fleukemia  with a spatients ,37. The e marrow . WT1 encllo-HSCT . The aut−3, down to 10−5. It is applicable for monitoring a variety of NPM1 mutations [DNMT3A and IDH mutations [Digital droplet PCR (ddPCR) is the new high-throughput technology that enables an absolute quantification of clonally amplified target genes without a reference standard amplification curve that is needed in conventional RQ-PCR. The ddPCR technique is characterized by high sensitivity exceeding 10utations , as wellutations  in AML autations ,51.NPM1 gene) and after applying error-correcting statistical models for raw sequencing data acquisition [DNMT3A, TET2, and ASXL1 (abbreviated as DTA) are known to be most frequent in CHIP, and thus may occur in healthy individuals with an increased incidence with age [NGS offers several advantages over PCR-based methods, such as the ability to study the entire leukemic genome, which can potentially be used in almost all AML patients. This molecular tool requires highly specialized bioinformatic approaches in order to identify all emerging clinically relevant variants or alleles. NGS used to detect MRD measures the variant allele frequency (VAF) of mutations. The sequencing approaches include Whole Genome Sequencing (WGS), Whole-Exome Sequencing (WES) and targeted-gene sequencing technology . The senuisition . Jongen-uisition . Howeveruisition ,55. Mutawith age ,56. Conswith age ,57. The NPM1-mutated AML treated with intensive chemotherapy may provide prognostic information useful in routine practice [NPM1-mutated transcripts after the second chemotherapy cycle in peripheral blood (PB) of patients in CR was the only significant prognostic factor. Notably as for the question of the timing of MRD assessment and the issue of bone marrow (BM) versus PB, assessment of MRD after the second chemotherapy cycle was more discriminatory than RT-qPCR positivity in PB at any other time point or assessing it in bone marrow samples. Moreover, the prognostic significance of the MRD status in the peripheral blood after the second chemotherapy cycle was also confirmed in the validation cohort. In fact, the presence of MRD compared to the absence of NPM1-mutated transcripts was associated with an increased 2-year cumulative incidence of relapse  and a lower OS rate . Additionally, MRD positive status after second cycle of chemotherapy in a subgroup of patients not harboring the FLT3-ITD mutation was also predictive for a very poor outcome with 2-year CI of relapse of 76% compared to 33% for MRD negative patients. Since the number of MRD positive patients who underwent HSCT in the first CR was small, the authors were not able to confirm a significant benefit of transplantation for those patients. This important question is currently addressed in an ongoing randomized phase III trial being conducted by the UK NCRI AML Working Group. The above UK NCRI AML findings are in line with the results of the Acute Leukemia French Association study, in which NPM1-mutated transcripts were assessed in PB after induction chemotherapy [NMP1 (<4-log) after chemotherapy was associated with poor outcome. Importantly, the study results indicated that HSCT improved OS and DFS only in patients with low reduction of NPM1. This finding is further supported by the results published by Freeman et al. demonstrating that in the subgroup of standard-risk patients without NPM1 mutations, only those with MRD+ benefit from HSCT in first CR [MRD assessment in the post-induction phase of the treatment of an AML patient is emerging as a new tool that can be used to predict poor outcome in standard-risk AML patients who may benefit from HSCT in the first CR. In this regard, the UK National Cancer Research Institute (UK NCRI) AML Working Group has evaluated prospectively the predictive value of MFC-MRD after first and second course of intensive chemotherapy in patients with AM . The patpractice . The resotherapy . Similarfirst CR .Regardless of the beneficial effect of allogeneic transplantation improving the outcomes of MRD-positive patients with low genetic risk, the results of HSCT in patients with intermediate and high-risk AML and positive MRD, for whom HSCT is the standard treatment approach, seem to be dismal, much worser than in patients with CR-MRD(−). Indeed, there is growing evidence confirming that also in AML, pre-transplant MRD assessment by either MFC or molecular techniques can be used for risk stratification at the time of HSCT following either myeloablative or reduced intensity conditioning ,62,63,64The molecular profiling and MRD assessment introduced into the prognostic stratification of patients with AML is a beginning of a new era of MRD-directed therapy, which will most probably allow patient-tailored and precision-type medicine avoiding under- or over-treatment of AML patients in the near future. In this regard, an important and critique question is the role of post-remission consolidation therapy in patients in CR with positive and negative MRD.The retrospective analyses of both the EBMT and CIBMTR registries strongly suggest no advantage of post-remission consolidation chemotherapy pre-transplantation with either MAC or RIC preparative regimens. Supporting the no role for post-remission consolidation chemotherapy are data published by Cahn et al. on behalf of the EBMT registry for AML patients undergoing HSCT from a matched related donors with MAC , and morAdditional important question is whether the kinetic of MRD clearance matter. Buccisano et al. analyzed the kinetics of reduction of MRD measured by MFC in bone marrow of AML patients treated with intensive chemotherapy according to the EORTC/GIMEMEA protocols . The autIn subsequent publication, Buccisano et al. addressed the question whether the patients in CR with positive MRD may benefit from allogeneic transplantation . In theiFLT3 internal tandem duplication (FLT3 ITD) inhibitors of FLT3, sorafenib, midostaurin, quizartinib, gilteritinib, and others, are also intensively assessed in active phase II–III trials [Because MRD emerges as a strong and independent risk factor for AML recurrence after allogeneic HSCT, a question arises regarding the prevention of post-transplant leukemia relapse in patients transplanted in CR-MRD(+) or in those with re-appearance of MRD after transplant. The increasing knowledge of both the molecular landscape of AML on one hand and better understanding of the biology of the GvL effect on the other opens the new perspectives on post-transplant MRD-targeted therapies, what has been very recently elegantly presented in expert review from the ALWP of the EBMT . PotentiI trials .In the context of suboptimal outcomes of HSCT in AML patients in complete remission but with positive MRD, the issues of whether selection of the type of graft or donor will improve results and whether the donor should differ in MRD positive as opposed to the MRD negative AML patient have been investigated. In other words, what is the optimal donor for MRD positive AML patients and whether alternative donors or mismatched donors with broader HLA disparities and thus theoretically stronger GVL are of advantage in eradicating MRD have been studied. Indeed, the impact of donor type in AML patients with pre-transplant MRD positivity was the subject of few retrospective studies also addressing the prognostic significance of MRD status for those type of transplantation. Specifically, the group from Seattle compared retrospectively the results of a first HSCT from an unrelated cord-blood (CB) donor versus transplantation from matched unrelated donor (MUD) or from an HLA-mismatched unrelated donor (mMUD) following myeloablative condition in a large group of patients with AML and myelodysplastic syndrome (MDS) who had MRD determined by MFC before transplantation . InteresThe molecular profiling and MRD assessment introduced into the prognostic stratification of patients with AML seem to begin a new era of MRD-directed therapy, which may allow avoiding under- or over-treatment of AML patients in the near future.WT1, mutated NPM1, t, inv(16), and t [WT1 analysis alone was with limited predictive value as well. Intriguingly, opposite results regarding the prognostic significance of testing the MRD status of the autograft by WT1 as the only tumor mutation were published by the group from Milan [WT1 expression level measured by RQ-PCR in autologous peripheral blood stem cells (PBSC) from leukapheresis discriminates the risk of relapse and is associated with DFS and OS after autologous HSCT.The development of highly sensitive and specific MRD assessment methods seems to lead to the rejuvenation of the concept of autologous transplantation in AML. Autologous transplantation can potentially allow a selected group of patients, especially with low and potentially intermediate genetic risk, to avoid over-treatment with allogeneic transplantation, providing 50–60% probability of long-term LFS . The rect . In the om Milan . They foIn conclusion, there is growing evidence that results of autologous HSCT in patients with low and intermediate risk AML are very good, provided that MRD status is negative before transplant.NPM1 mutation who are MRD positive after the second course of chemotherapy should be considered for early allogeneic transplantation. As for the role of pre-transplant consolidation, there are still controversies about its necessity in patients that achieved remission but are MRD positive and are about to go allogeneic transplantation, since there is no evidence that consolidation can eradicate MRD in AML. On the other hand, there are strong data regarding the independent impact of pre-transplant MRD status on the outcomes of HSCT, with similar significance whether MAC and RIC were applied. The broad availability and predictive capabilities of MRD as summarized in the current review may open new perspectives for novel targeted therapies both for pre-transplant MRD eradication as well as for post-transplant maintenance therapy in both ALL and AML. Lastly, as for the question of optimal selection of donor depending on AML patients’ MRD status, the published data, although on limited number of patients, may suggest that the negative impact of MRD on transplant outcomes might be partially overcome by allotransplant from cord blood graft or haploidentical donors in comparison to transplantation from unrelated or sibling donors. However, due to small size of study group and retrospective nature of the analysis these somewhat provocative data should be confirmed in further studies.Measurable residual disease has been used to guide therapeutic decisions and therapies in pediatric acute lymphoblastic leukemia for more than two decades. The lessons learnt from pediatric ALL were gradually incorporated into adult ALL and AML therapeutic strategies. Data are currently emerging for both adult ALL and AML that, similar to pediatric ALL, MRD is a new, extremely important prognostic factor in both types of adult acute leukemia. MRD should be most probably incorporated in the treatment decision process for both adult ALL and AML, similar to being used in pediatric ALL, although there are still many open questions and some controversies discussed in our review. The widely available MFC and RQ-PCR methods for MRD measurement may allow the introduction of MRD into the risk stratification in AML in daily clinical practice in the nearest future. It may lead to treatment de-escalation in patients with intermediate genetic risk who achieve CR with MRD negativity during the post-remission therapy. The era of MRD-directed therapy in AML may even lead to revisiting and new perspectives for autologous transplantation for patients with low or even intermediate risk AML achieving MRD negative status, although this approach is controversial as GVL is of major importance in curing AML. On the other hand, there is strong evidence that patients with