To assess the Nickel sensitizing potential of total knee arthroplasty (TKA), explore the relationship between hypersensitivity and clinical outcomes, and evaluate the utility of skin patch testing pre- and/or postoperatively.A literature search was performed through EMBASE, Medline and PubMed databases. Articles were screened independently by two investigators. The level of evidence of studies was assessed using the Oxford Centre for Evidence-Based Medicine Criteria and the quality evaluated using the Methodological Index for Non-randomized Studies and Cochrane risk-of-bias tools.Twenty studies met the eligibility criteria, reporting on 1354 knee arthroplasties. Studies included patients undergoing primary or revision TKA, pre- and/or postoperatively, and used patch testing to identify Nickel hypersensitivity. Prevalence of Nickel hypersensitivity ranged from 0% to 87.5%. One study compared the prevalence of Nickel hypersensitivity in the same patient group before and after surgery and noted newly positive patch test reactions in three patients (4.2%). Three studies reported lower prevalence of Nickel hypersensitivity in postoperative patients compared to preoperative ones. Seven studies suggested that hypersensitivity might cause adverse clinical outcomes, but six did not support any relationship. Seven studies recommended preoperative patch testing in patients with history of metal allergy, and nine concluded that testing may be valuable postoperatively.Patients undergoing TKA with no prior history of metal hypersensitivity do not seem to be at an increased risk of developing Nickel hypersensitivity, and there is conflicting evidence that patients with pre-existing hypersensitivity are more likely to experience adverse outcomes. Patch testing remains the most commonly used method for diagnosing hypersensitivity, and evidence suggests preoperative testing in patients with history of metal allergy to aid prosthesis selection, and postoperatively in patients with suspected hypersensitivity once common causes of implant failure have been excluded, since revision with hypoallergenic implants may alleviate symptoms.The online version contains supplementary material available at 10.1186/s42836-022-00144-5. The reported prevalence of metal hypersensitivity in the general population ranges from 10 to 15%  and in cPatients with metal hypersensitivity can present in a similar way to joint infection , 8. SympMetal hypersensitivity is a diagnosis of exclusion once more common causes of implant failure, such as infection and aseptic loosening, have been ruled out , 13. CurSince metal hypersensitivity occurs most frequently from exposure to Nickel, this systematic review was performed to collate and analyze the current literature on Nickel hypersensitivity in patients undergoing TKA. Previous review articles , 13–19 hThis review was conducted in accordance with the 2020 Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines [Clinical studies determining the prevalence of Nickel hypersensitivity by patch testing patients with total knee arthroplasty, pre- and/or postoperatively, were included. Studies which also involved patients undergoing orthopedic interventions other than TKA were not excluded, provided that an appropriate number of TKA patients were included. Full-text articles had to be available and published in English or with translation freely available. Case reports, review articles, conference abstracts and surveys were excluded.A comprehensive electronic search strategy utilizing a combination of Medical Subject Heading (MeSH)-terms and keywords was developed by one author (CP) and refined with the help of the Department Librarian (PA). The EMBASE and Medline databases were searched using the Healthcare Database Advance Search (HDAS) platform and extended to the native PubMed database, identifying literature from inception until September 2021. The line-by-line strategy run in HDAS and PubMed is outlined in Supplementary Material To supplement the electronic search, a detailed review of the reference lists of the final studies included in the systematic review and in review articles on the same or similar topic was performed. Finally, a search of the grey literature on OpenGrey was performed to identify any published or ongoing research.Potentially eligible studies were identified by screening the titles and abstracts of all articles retrieved from the search. The eligibility of each full-text article was then assessed for inclusion. Each stage was performed independently by two investigators , and any inconsistencies were discussed until consensus obtained. Disagreements at either stage were resolved by the senior author (DHS).e.g. author, year, country, etc.)Study characteristics Patient characteristics Type of TKA implant Details of patch testing Prevalence of Nickel hypersensitivity Relevant clinical results (Main conclusions and recommendationsThe following data were extracted from the included studies:A data collection table in Microsoft Excel was designed by one author (CP) to display the information extracted from each eligible study.Owing to heterogeneity in study design, participants, interventions and outcome measures, a quantitative meta-analysis was not appropriate.Levels of evidence (LE) were assessed using the Oxford Centre for Evidence-Based Medicine (OCEBM) framework .The quality of observational studies was independently assessed by two authors  using the Methodological Index for Non-randomized Studies (MINORS) tool . The ideAny randomized controlled trials were scored using the revised Cochrane risk-of-bias tool for randomized trials (RoB 2) .The assessments provided an overall impression of each study but were not used to weight any studies in the analysis.The initial search performed through the EMBASE, Medline and PubMed databases identified 4002 records, of which 1695 records remained after deduplication. Of these, 1666 were excluded after screening titles and abstracts. Eight additional studies were identified by searching the reference lists of articles on the same topic, and no studies were identified by performing a search of the grey literature. As a result, 37 full-text articles were assessed for eligibility for inclusion, of which 17 were excluded e.g. no implant) or undergoing a different surgical procedure, such as THA. Fifteen studies [All patients underwent primary or revision TKA, with the other study participants comprising either control groups , ceramic implants (oxidized Zirconium), pure Titanium implants, and all-polyethylene tibial components , 47. Sate.g. with topical steroids or NSAIDs [The evidence suggests that patch testing could be a valuable diagnostic tool postoperatively to screen for metal hypersensitivity in symptomatic patients following TKA. In patients presenting with recent onset of periprosthetic dermatitis, arthralgia, evidence of loosening, or radiolucent lines on radiographs, patch testing seems a reasonable option once other failure mechanisms such as infection, instability and malalignment have been excluded and inflammatory markers (CRP and ESR) and joint aspiration have yielded negative results , 14. A tr NSAIDs ) or consLymphocyte transformation testing (LTT) was performed in addition to patch testing in a number of studies included in the review , 37, 41.Patch testing remains the most commonly used investigation for diagnosing metal hypersensitivity , 14, 49.This systematic review has several limitations. Firstly, all the included studies had low levels of evidence, with only one  scoring Several of the articles analyzed groups of patients undergoing not only knee, but also other orthopedic interventions , 37, 41,e.g. six months) may be insufficient to detect new hypersensitivity reactions to implant components [The utility of late patch test reading at day six after application has been documented  and it imponents .The current literature does not support the concept that patients undergoing TKA with no prior history of Nickel hypersensitivity are at an increased risk of developing hypersensitivity, and there is conflicting evidence that patients with established Nickel hypersensitivity are more likely to experience dermatological or orthopedic complications such as persistent pain, implant loosening or failure. Despite its limitations, cutaneous patch testing remains the most commonly used method for diagnosing Nickel hypersensitivity. The literature does not support routine patch testing of patients prior to TKA but does support performing this test in patients with a history of metal hypersensitivity. In those with a positive patch test, the choice of implant to use should be made on a case-by-case basis after discussion with the patient, as in the absence of more robust evidence, the careful selection of which device to implant may minimize the potential risk of complications related to metal hypersensitivity. Patients with a clinical presentation suggestive of Nickel hypersensitivity following TKA may benefit from patch testing only after the more common causes of pain, loosening and failure have been excluded, since revision surgery with hypoallergenic implants may alleviate symptoms. To further establish the relationship and importance of Nickel hypersensitivity in patients undergoing TKA, large-scale, appropriately designed studies will be required.Additional file 1. Search strategy used in HDAS.Additional file 2. Search strategy used in PubMed.


Engineering modified extracellular vesicles is an emerging component of bone tissue engineering and its main progression and clinical applications will be discussed. Finally, future perspectives and challenges of developing extracellular vesicle-based regenerative medicine will be given. This review may provide a theoretical basis for the future development of extracellular vesicle-based biomedicine and provide clinical references for promoting the repair of critical-size bone defects.In recent years, the incidence of critical-size bone defects has significantly increased. Critical-size bone defects seriously affect patients’ motor functions and quality of life and increase the need for additional clinical treatments. Bone tissue engineering (BTE) has made great progress in repairing critical-size bone defects. As one of the main components of bone tissue engineering, stem cell-based therapy is considered a potential effective strategy to regenerate bone tissues. However, there are some disadvantages including phenotypic changes, immune rejection, potential tumorigenicity, low homing efficiency and cell survival rate that restrict its wider clinical applications. Evidence has shown that the positive biological effects of stem cells on tissue repair are largely mediated through paracrine action by nanostructured extracellular vesicles (EVs), which may overcome the limitations of traditional stem cell-based treatments. In addition to stem cell-derived extracellular vesicles, the potential therapeutic roles of nonstem cell-derived extracellular vesicles in critical-size bone defect repair have also attracted attention from scholars in recent years. Currently, the development of extracellular vesicles-mediated cell-free regenerative medicine is still in the preliminary stage, and the specific mechanisms remain elusive. Herein, the authors first review the research progress and possible mechanisms of extracellular vesicles combined with bone tissue engineering scaffolds to promote bone regeneration  Critical-size bone defects refer to the smallest intraosseous wound that cannot heal spontaneously . The aetin vitro expansion, culture conditions, cell transplantation procedures and recipient pathological microenvironments also have adverse effects on the survival and biological characteristics of cells (Bone tissue engineering (BTE), which uses biomaterials with sophisticated biophysical or biochemical features to circumvent the constraints described above, has made great progress in repairing critical-size bone defects. BTE includes three basic components: biological scaffolds, seed cells and bone inductive factors . Biologiof cells . Many stof cells , but keyIn recent years, cell-free regenerative medicine for critical-size bone defects has received extensive attention . Evidencvia bioactive molecules. Engineering modified EVs is an emerging component of BTE. Their main progress and opportunities will then be discussed. Finally, future perspectives and challenges of the developing EV-based regenerative medicine are given. Thus, this review may provide a theoretical basis for the future development of the EV-based biomedicine and provide clinical references for promoting the repair of critical-size bone defects.Although considerable research on EVs has focused on promoting the repair of critical-size bone defects, their development is still in the preliminary stage, and the specific mechanisms remain elusive. What is more, for critical-size bone defects, application of EVs alone could not fill defect spaces and guide bioactive molecules to repair and reconstruct the bone defect. Thus, some bioactive scaffolds are needed in EVs-based BTE application. Herein, the authors first review the research progress and possible mechanisms of EVs combined with BTE scaffolds to promote bone regeneration in vivo. To date, multiple studies have demonstrated that very few transplanted cells can be tracked in vivo after 4–8 weeks, suggesting that their regenerative ability is more likely to be associated with the indirect mechanisms of seed cells, including cytokine paracrine, immunoregulation, and signal transduction -derived EVs were demonstrated to have a good osteogenic inductive effect  and osteopontin along with increased levels of the vascular marker CD31 both in healthy  scaffolds with a polydopamine coating. ct sites . Scholarct sites . Polycap promoting osteogenic differentiation of stromal cells while inhibiting the differentiation of osteoclasts; 2) enhancing angiogenesis to provide a suitable environment for bone regeneration; and 3) modulating inflammatory responses to maintain a favourable immune microenvironment . Thus, EAlthough natural EVs exert diverse functions in regenerative medicine through multiple biological mechanisms, their limitations in repairing critical-size bone defects have gradually been recognized. Specifically, the targeting effect of natural EVs is poor. Most of the mature EVs delivered to bone defect sites will enter the circulatory system and cannot all accurately act on the target cells. In addition, not all EVs isolated from parent cells have therapeutic effects; thus, the number of effective natural EVs is far from sufficient. As a result, the bioactive molecules that are needed in different pathological circumstances cannot reach a therapeutic concentration. Although EVs are processed and assembled in parent cells and inherit similar biological characteristics to them, the bioactive molecules in natural EVs vary greatly, which will bring unstable efficacy in repairing critical-size bone defects. Therefore, engineering-modified EVs have been investigated to accelerate the bone regeneration process by applying multiple techniques that fall into two main categories in most circumstances: endogenous engineering strategies and exogenous engineering strategies. Advantages and disadvantages of some commonly used techniques are summarized in .In the previous sections, the authors summarized the biological characteristics of EVs derived from different parent cells and their applications in BTE to repair critical-size bone defects. Studies have shown that except for selecting appropriate types of parent cells, modification of the parent cells before EV isolation through genetic strategies, preconditioning treatment and physical manipulations could also modify the biological characteristics of EVs . These aRUNX2 gene, the osteogenic differentiation of hBMMSCs was potentiated. Meanwhile, the differentiation induction capabilities of EVs derived from this gene-transfected cell were also enhanced in vitro (in vitro results indicated that EVs from HIF-1α-upregulated MSCs could better enhance matrix mineralization, ALP activity and osteogenesis-related gene expression in BMMSCs and promote the migration, proliferation and tube formation of HUVECs. Meanwhile, in vivo results indicated better bone tissue regeneration and neovascularization; therefore, the trabecula bone healing time was significantly shortened in rabbit necrotic femoral heads (Genetically modifying the natural procedures of protein synthesis of parent cells can endow the modified EVs with membrane modification and more desirable functions, such as precise targeting and individualized therapeutic performance, by regulating some targeted biomolecules. Compared with natural EVs, specific ligands can be added to the EV membrane to improve its targeting effects through genetic engineering . After tin vitro . In a sial heads . Anotheral heads . Other ral heads . They obal heads .in vitro and of promoting neovascularization in 3D cultures in vivo. in vivo. Another study found that exosomes derived from hBMMSCs preconditioned with dimethyloxaloylglycine, which stabilizes HIF-1α at a low concentration, could improve bone healing by boosting angiogenesis in critical-sized calvarial defects in rats (Preconditioning treatment of parent cells through conditioned culturing medium could improve the biological activities of EVs derived from them. It is an effective strategy to boost EV-mediated BTE in regenerating bone tissues. Preconditioning treatment can directly modify the DNA or RNA of the parent cells to obtain needed EVs rich in target cargoes.  in rats . Moreove in rats . The obtin vitro experiments showed that these modified EVs could promote the proliferation and migration of HUVECs and tube formation. Due to the favourable angiogenic microenvironment, osteogenic differentiation of BMMSCs at the bone defect site was also enhanced, which was manifested by increased gene expression of serum OCN and ALP (in vivo experiments were performed using the steroid-induced avascular necrosis model in the femoral head. The obtained results indicated that the femoral head transplanted into EVs derived from hypoxia-preconditioned cells had higher vascular density and denser trabecular bone tissue formation (in vivo applications of modified EVs showed more favourable new bone formation.Another widely applied preconditioning treatment of parent cells is a hypoxic environment. MSCs, which were preconditioned in a hypoxic environment, promoted angiogenesis capability. In addition, EVs derived from these cells also had better angiogenic characteristics and led to enhanced osteogenic capability. The  and ALP . The in ormation . The resPhysical manipulation could change the amount of EVs released from parent cells together with the type and amount of therapeutic cargoes in EVs. Generally, physical manipulation is performed by directly exerting physical forces on the parent cells by electroporation and extrusion , therebyTo summarize briefly, exogenous engineering of EVs involves functional modification after their isolation, directly loading specific external cargoes into nanoscale EVs to deliver bioactive molecules with therapeutic effects to the local microenvironment, thereby promoting critical-size bone defect repair . Currentin vitro. In addition, demineralized bone matrix loaded with these novel complexes exhibited increased bone regeneration induction capability after being implanted subcutaneously in rat and rabbit femoral head bone defect models.VEGF plays a critical role in angiogenesis and BTE for bone tissue regeneration . HoweverSHN3 gene to enhance their therapeutic effects. Modification of a bone-targeting peptide endowed the engineered exosomes an ability to deliver siRNA to osteoblasts specifically. Silencing of the osteoblastic SHN3 gene enhanced osteogenic differentiation, and inhibited osteoclast formation. Furthermore, SHN3 gene silencing could facilitated vascularization, especially formation of type H vessels. This study demonstrated that exogenous engineered exosomes could serve as a promising therapy vesicle to kill three birds with one stone. With meticulous and thorough research exploring the biogenesis and biofunctions of EVs, natural and engineered modified EVs that possess various promising advantages, such as biological functions similar to those of parent cells, low immunorejection, desirable biocompatibility, and the capability to deliver individualized drugs, have shown exciting application prospects in diagnosing and treating diverse pathological conditions . The firAlthough extensive studies have focused on various therapeutic mechanisms of natural or engineered modified EVs for different diseases, such as cancer and inflammatory diseases , the potin vivo application of EVs cannot be separated from the scaffold materials. In current studies, based on the combined applications of different scaffold materials, scholars have developed a variety of innovative methods to improve the delivery efficiency of EVs (Future studies at this stage will mainly focus on exploring the mechanisms of inducing bone regeneration and manufacturing more ideal scaffold materials. Theoretically, EVs regulate bone defect repair and regeneration through the interaction of their multiple internal cargoes . Howevery of EVs . Howevery of EVs . In the in vivo bioeffects of EVs, such as distribution or bioelimination in blood circulation or in diverse pathological microenvironments, will also provide a deeper understanding of the pharmacokinetic and pharmacodynamic performances of EVs. This broader understanding will be beneficial to the proposal of EV-mediated targeted nanotherapeutic strategies.Furthermore, deeper investigations of As reviewed earlier, EVs have more favourable biocompatibility and enhanced biosafety over conventional stem cell therapy; however, the whole components of EVs, including some other unknown bioactive molecules, are still not completely clear . AlthougCurrently, the optimal therapeutic dose of EVs is still uncertain; thus, there would also be potential side effects after application. More preclinical studies are needed, especially on applying EVs in bone defect repair in large animals, to provide more safety references and a basis for the clinical promotion of EVs in later stages.Effective isolation of EVs is the basis for their clinical application. From this point of view, standardized purification, preparation, production and preservation are essential. Increasing evidence indicates that EVs from different types of parent cells carry distinct bioactive molecules that play different biological functions in bone remodelling and regeneration . Even EVCurrently, differential ultracentrifugation and sequential ultrafiltration are the most commonly employed approaches to isolate EVs . HoweverAccordingly, technology to accurately regulate the expression of bioactive molecules in EVs is crucial to achieve more desirable tissue repair. Therefore, we will further explore the engineering strategies of EVs through gene modification, specific preconditioning treatment, external gene/protein loading or combination to prepare EVs with enhanced bone induction ability. In addition to engineering modification of EVs, improving the loading efficiency of internal cargoes without interfering with the natural lipid and protein compositions of EVs is also an important issue that cannot be ignored.Critical-size bone defects have become a serious threat to populations at both the individual and societal levels. The progress of stem cell therapy has led to promising results for remodelling and regeneration of bone tissues. However, stem cell therapy has some disadvantages that restrict its wider clinical applications. These disadvantages, together with problems caused by storage and transport, further limit the application of MSCs in repairing critical-size bone defects. An increasing amount of convincing evidence has demonstrated that most beneficial functions of stem cells are attributed to EVs, which gives rise to a new paradigm in which EVs secreted from both stem cells and other cells promote bone regeneration by modulating crosstalk between EVs and resident bone cells or the surrounding microenvironment. Although the utilization of EVs as bioactive nanotherapeutics in regenerative medicine has just started from the preliminary stage, the results of preclinical tests showed that applications of EVs would overcome limitations of stem cell-based therapies and possible side effects associated with the administration of stem cells. In addition, some unique advantages of EVs, including low immune rejection, feasibility to modify membrane constituents, ability to pass the blood‒brain barrier, convenient isolation, and lack of tumorigenicity, make the application of EVs a promising choice in biomedical treatment for critical-size bone defects.ex vivo do not exactly mirror those extracted in vivo when parent cells are exposed to specific diseases or injury-related conditions.Accumulated findings suggest that EV-mediated therapy may be an effective alternative to regulate the bone metabolism microenvironment. Here, the authors reviewed the molecular mechanisms of the effects of EVs on osteogenesis, osteoclast differentiation, angiogenesis, and immune reactions. It is well-recognized that EVs derived from different sources have different internal cargoes, such as inflammatory cytokines, signalling molecules, mRNAs, miRNAs and lncRNAs. The diverse capabilities of EVs come from different internal cargoes. Currently, scholars mainly focus on miRNAs, which play a critical role in EV-mediated critical-size bone defect regeneration. However, researchers had no information about critical proteins and other RNAs involved in the interactions between different EVs and EV-target cells, especially in distinct pathological circumstances. These problems must be of great interest to future preclinical and clinical studies aiming to explore the underlying therapeutic mechanisms of EVs. In addition to mechanistic studies, further functional studies of EV-mediated therapy are also needed, as EVs extracted Efficient delivery, bioactivity maintenance, and controlled/sustained release will become key factors for the safe and effective application of EVs in critical-size bone defect repair. Before resolving these issues, the isolation and characterization techniques need to be standardized first, but the field has yet to achieve consensus. Although scholars in EV-mediated BTE face numerous challenges, with continued advances in understanding EV biology and therapeutic mechanisms, together with the rapid development of related disciplines, such as bioengineering, chemistry, material science, and nanotechnology, it is believed that EVs will stimulate further explorations and inspirations for tissue engineering and efficient nanotherapeutics in the near future, eventually bringing the spring of cell-free regenerative medicine for critical-size bone defect repair.


Menopause transition can be interpreted as a vulnerable state characterized by estrogen deficiency with detrimental systemic effects as the low-grade chronic inflammation that appears with aging and partly explains age-related disorders as cancer, diabetes mellitus and increased risk of cognitive impairment. Over the course of a lifetime, estrogen produces several beneficial effects in healthy neurological tissues as well as cardioprotective effects, and anti-inflammatory effects. However, clinical evidence on the efficacy of hormone treatment in menopausal women has failed to confirm the benefit reported in observational studies. Unambiguously, enhanced verbal memory is the most robust finding from longitudinal and cross-sectional studies, what merits consideration for future studies aiming to determine estrogen neuroprotective efficacy. Estrogen related brain activity and functional connectivity remain, however, unexplored. In this context, the resting state paradigm may provide valuable information about reproductive aging and hormonal treatment effects, and their relationship with brain imaging of functional connectivity may be key to understand and anticipate estrogen cognitive protective effects. To go in-depth into the molecular and cellular mechanisms underlying rapid-to-long lasting protective effects of estrogen, we will provide a comprehensive review of cognitive tasks used in animal studies to evaluate the effect of hormone treatment on cognitive performance and discuss about the tasks best suited to the demonstration of clinically significant differences in cognitive performance to be applied in human studies. Eventually, we will focus on studies evaluating the DMN activity and responsiveness to pharmacological stimulation in humans. Menopause is a biological milestone linked to the onset of cognitive impairment, amongst several deleterious systemic effects. There would be a window of opportunity to provide hormone therapy (HT) that would benefit users. Clinical studies show cognitive improvements mainly in verbal memory Maki,  in womenClinical studies have revealed higher activation of fronto-cingulate regions in menopausal women under estrogen treatment by brain functional magnetic resonance imaging (fMRI), although in many cases, no difference in cognitive performance was demonstrated. fMRI studies may also have different appreciations, thus the abnormal activation observed in some individuals who executed adequately the imposed task could be interpreted as a compensatory activation due to sub-clinical cognitive impairment   within 2–4 weeks  than middle-aged rats, since E2 synthesis  produces a gradual loss of ovarian follicles and EIn 3-month-old rats, icv streptozotocin (STZ) produces similar memory and learning alterations to those observed in animal models of Alzheimer's disease (AD) , found at synapses and presynaptic terminals in neurons in both male and female brains of rodents, monkeys, birds, amphibians, and humans. NDE2 regulates sexual differentiation, reproduction, synaptic plasticity, cognition, neuroinflammation, and neuroprotection  and never users of oral contraceptive pills (n = 33), and their DMN connectivity and attention networks were recorded by fMRI. E2 was beneficial to modulate attention and conscious processes promoting normal fear extinction learning and extinction memory retention. This kind of modulation by exogenous E2 has not been related to a unique network modulation, but to an activation of whole-brain functional connectivity, implying functionally distinct systems as the anterior component of DMN, i.e., regions with high levels of E2 receptors  (Ramírez-Barrantes et al., 2 receptors, such as TRPV1, might be critical for the maintenance of intracerebral functional connectivity during acute E2 stimulation. Future clinical trials focused on these rapid signal changes following estrogen exposure may identify predictors of long-lasting HT effectiveness.Different timescales underlie E effects . GenomicIM and PO conceived the idea and wrote the manuscript. SC, JM-P, RS-Z, and RR-B developed contents in rs-fMRI, animal models, connection with human studies, and DMN, respectively. LA reviewed critically neurological topics. CC developed figures. All authors reviewed critically the final version for intellectual contents.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Endophytic bacteria inhabiting plant tissues are generally considered to play a crucial role in host adaptation to biotic stresses and adverse environmental conditions . In receThis Research Topic aims to present new research results focusing on the role of endophytic bacteria and the complex interactions they establish with plants to improve host resistance to stress under different environmental conditions.Mutungi et al. showed colonization of common bean (Phaseolus vulgaris) by endophytic strains of Bacillus megaterium and Enterobacter hormaechei, respectively isolated from the perennial herbs Boeravia erecta and Abutilon fruticosum, promoted plant growth and reduced the severity of the disease caused by infection with Fusarium solani. The growth inhibition of F. solani was also confirmed by co-culturing with these endophytes in vitro. Both strains possessed traditional plant growth-promoting traits for the production of phytohormone indole acetic acid (IAA) and solubilization of phosphates. Similarly, Gupta et al. reported that endophytic strains of Pseudomonas aeruginosa and Aneurinibacillus aneurinilyticus, isolated from “tulsi” (Ocimum tenuiflorum) plants, could induce ISR in pea (Pisum sativum) when challenged with pathogenic Fusarium wilt (Fusarium oxysporum f.sp. pisi). It was hypothesized that these endophytes could exert their antagonistic and growth-promoting effects by releasing secondary metabolites, including volatile organic compounds (VOCs), siderophores, and ammonia, or by mobilizing hitherto inaccessible inorganic nutrients.Endophytic bacteria produce diverse metabolites that may benefit the plant hosts in the growth promotion and control of diseases . Many diStreptomyces lincolnensis, isolated from wormwood (Artemisia annua), to regulate the expression of genes controlling root morphogenesis in Arabidopsis thaliana .Interestingly, the involvement of endophytic traits for phosphate solubilization, iron acquisition, ACC deamination, nitrogen fixation, and IAA production was shown to underpin the mechanism used by Controlling the uptake and bioavailability of micronutrient, mineral, and trace element levels in plants plays an essential role in plant growth and development. Fine regulation of these steps also involves interaction between the plant host and its microbial colonizers . MoreoveBrevibacillus brevis  rhizosphere to understand whether neighboring maize crops could alter root microbiome assembly. They found that maize coexistence re-shaped peanut-associated belowground microbial communities and induced colonization by plant-growth-promoting rhizobacteria, such as Bradyrhizobium sp. and Streptomyces sp., with relevant potential for effective nutrient accumulation and phytohormone production.nifH) to enhance nitrogen fixation. Taken together, these findings reinforce the hypothesis that plants establish a plant-microbial holobiont through a selective co-evolution with their endophytic inhabitants (Similarly, abitants  and thisModern sustainable agriculture is nowadays looking at this “plant holobiont breeding” approach  which atBringing together seven original research papers, this Research Topic provides a state-of-the-art overview of the beneficial effect that bacterial endophytes may exert on plants from different perspectives, with special emphasis on their ability to increase the potential of host plants to tolerate biotic and abiotic stresses.We regard this collective effort as an important opportunity to recall the importance of directing future research on plant endophytes towards an in-depth analysis of their modes of interaction with hosts. Based on this approach, rapid progress could be made in the discovery of natural plant growth promoters and microbial fertilizers to provide sustainable alternatives to the massive use of synthetic pesticides, hindered by the “Green Deal” innovations . We hopeYL and MM wrote the first draft of the manuscript. X-XZ, JK, SQ and Y-HZ edited the manuscript and provided a critical review. X-XZ submitted the final version. All authors made a substantial, direct and intellectual contribution to the work, and approved the submitted version.


The vagus nerve is involved in the autonomic regulation of physiological homeostasis, through vast innervation of cervical, thoracic and abdominal visceral organs. Stimulation of the vagus with bioelectronic devices represents a therapeutic opportunity for several disorders implicating the autonomic nervous system and affecting different organs. During clinical translation, vagus stimulation therapies may benefit from a precision medicine approach, in which stimulation accommodates individual variability due to nerve anatomy, nerve-electrode interface or disease state and aims at eliciting therapeutic effects in targeted organs, while minimally affecting non-targeted organs. In this review, we discuss the anatomical and physiological basis for precision neuromodulation of the vagus at the level of nerve fibers, fascicles, branches and innervated organs. We then discuss different strategies for precision vagus neuromodulation, including fascicle- or fiber-selective cervical vagus nerve stimulation, stimulation of vagal branches near the end-organs, and ultrasound stimulation of vagus terminals at the end-organs themselves. Finally, we summarize targets for vagus neuromodulation in neurological, cardiovascular and gastrointestinal disorders and suggest potential precision neuromodulation strategies that could form the basis for effective and safe therapies. The vagus is the tenth and longest cranial nerve which brings the brain and internal organs in bidirectional communication  and posteriorly  to the esophagus, known as anterior and posterior trunks of the vagus Ellis . The antThe liver is innervated by the hepatic branch from the anterior vagal trunk  pulses , uses frequently alternating (> 1 kHz) rectangular or sinusoidal current to induce reversible neuromodulation. The majority of studies investigated inhibitory effects that associate with neural conduction block , such as epilepsy and neuroinflammatory related diseases, as it carries somatosensory signals from the ear with the same projection at NTS. With its superficial nerve ending near the ear (tragus and auricle), the auricular vagus nerve stimulation (aVNS) can be delivered percutaneously or transcutaneously, offering a method to modulate neural activity on the vagus nerve with the potential for a more favorable safety profile. Given aVNS can be implemented with minimally invasive approaches and has the potential to modulate vagal activity, there have been many early-stage clinical trials investigating a diverse range of potential therapeutic indications, including heart failure, epilepsy, depression, pre-diabetes, Parkinson’s, and rheumatoid arthritis, and several aVNS devices were already developed for various applications  devices, such as GammaCore , are designed primarily to stimulate myelinated sensory afferent vagus nerve fibers as they ascend through the neck in the carotid sheath, using a battery-powered external electrical stimulator. This device has been approved and is being prescribed in several countries mainly for the treatment of primary headache and is CE marked in the EU for the treatment of primary headache, epilepsy, bronchoconstriction, anxiety, depression, and gastric motility disorders . The three largest clinical trials of cervical VNS  in patients with HFrEF on top of optimal medical treatment yielded in mixed results . Recently, two randomized and double-blind clinical trials showed that LLTS reduces TNF-a, improves left ventricular strain and quality of life in this population  inducibility and duration. These findings were also consistent with an LLTS study in canines. Notably, LLTS reduces pacing-induced AF burden and inflammatory cytokines in humans with paroxysmal AF at the electrophysiology lab and also in a pilot randomized sham-controlled trial with chronic low-level tragus stimulation in patients with paroxysmal AF, LLTS resulted in 85% lower AF burden at 6 months along with 23% lower TNF-α levels compared to the sham group.The vagus nerve (VN) innervates all the major gastrointestinal (GI) organs in the abdominal cavity. Afferent and efferent nerves within the branches of the VN enter and leave the GI organs. Selective stimulation of the VN innervating GI organs can be accomplished by all three strategies, either at the cervical level, at near-organ branch level, and on-organ through ultrasound stimulation. Here, we discuss studies related to GI disorders that use or could benefit from using prevision vagus neuromodulation.Recently, VNS was proposed to treat inflammatory bowel disease, based on its ability to suppress inflammation through inflammatory reflexes. Several animal studies have demonstrated the anti-inflammatory effects of cervical VNS on animal models of IBD  is a common disorder in patients undergoing abdominal surgery. The pathogenesis of POI is thought to be due to cytokine release during and after the surgery. TNF-α released from inflammatory muscular lamina propria and activated permanent macrophages  or control (n = 102) group  had a mean excess weight loss (EWL) of 21% ; 58% of participants had ≥5% TWL and 34% had ≥10% TWL. Among the subset of participants with abnormal preoperative values, significant improvements were observed in mean LDL and HDL cholesterol, triglycerides, HbA1c, and systolic and diastolic blood pressures. Heartburn/dyspepsia and implant site pain were the most frequently reported adverse events. The primary related serious adverse event rate was 4.3%. It was concluded that vBloc therapy continues to result in medically meaningful weight loss with a favorable safety profile through 2 years. Finally, Huerta et al. has reported the use of ultrasound stimulation at the portahepatis in the mice model of obesity  of 30 kg/mVagus nerve is a promising bioelectronic target for many chronic disorders; however, precision neuromodulation is needed to expand the horizon of the VNS application. Considering its anatomical structure, the vagus nerve can be stimulated at three different locations. First, at the cervical level, developing the nerve-electrode interface that preferentially stimulates a part of the fascicular structure, and stimulation strategies that can engage specific fiber types will give rise to the possibility of precision VNS that targets specific functions. Second, an electrode implanted on the individual branch that is near the target organ to effectively modulate specific neural signaling pathways. Third, at the organ level with the use of non-invasive ultrasound. We believe the precision VNS can be achieved through one of the above strategies, and the optimal approach should be highly disease-oriented and anatomically driven.


Plant-microbe interactions in natural and agricultural ecosystems have attracted more attention than ever , harnessing repeatable and applicable benefits of microbes is definitely still in its infancy. Repeatability and deterministic processes could be observed under well-controlled experimental conditions, such as using a synthetic community (SynCom) of microbes  symbiosis—symbionts of plants such as mycorrhizal fungi and rhizobia assist in nutrient acquisition and plant growth improvement , breeding, etc., are challenges for sustainable agriculture. The current Research Topic collects 24 papers that consider, among others, fertilization, continuous cropping, and grazing.Gu et al. found that N application indeed changed bacterial diversity and community structures of sugarcane cropping systems. Excessive application of N fertilizers had unexpectedly led to a lower yield. This also echoes Chen J. et al.'s view that an optimized level of chemical input is needed to keep useful microbe functioning.To improve crop yield, applying nitrogen (N) fertilizer in soils is an efficient approach. However, N input and overuse create N emission/leaching problems . For instance, Yang et al. reported that long-term (up to 30 years) alfalfa cultivation in the field enhanced soil microbial diversity and bacterial networking, higher than those short-term cultivations of meadows.Apart from fertilization, continuous cropping is common in agriculture. Continuously planting the same or similar cultivars in the same soils often leads to plant growth inhibition and serious soil-borne diseases, which is known as continuous cropping obstacle (CCO) Shipton, . The imbWang et al. show that the 4-year grazing exclusion did not increase the complexity and connectivity of bacterial co-occurrence patterns indicating a longer term of grazing exclusion is needed for soil restoration in terms of microbial activities. The optimal duration of grazing exclusion seems essential for the restoration depending on the type of ecosystem  and attacked by herbivore pests  triggered by plant growth-promoting rhizobacteria (PGPR) can resist herbivores. Herbivores can also induce plant volatile production. Understanding such chemical-mediated plant-insect interactions is important to achieve sustainable agriculture. Cheng et al. review the roles of arbuscular mycorrhizal (AM) fungi (ancient and widespread symbiotic fungi of land plants) in helping their plant hosts to resist drought stress. Cheng et al. discuss the AM fungal diversity and activity, symbiotic relationship, morphological, physiological, and molecular mechanisms of AM fungi in assisting plant drought resistance. An outlook for future research is also provided. Not only fungi but also bacteria were considered to resist drought stress. Armanhi et al. study the impact of a synthetic community (SynCom) on the physiology and response of maize under drought stress. Results suggest SynCom inoculation reduced biomass loss and modulated vital physiological traits, such as lower leaf temperature, reduced turgor loss, and faster recovery upon rehydration. Gebauer et al. also determined the effect of water deficit history on soil microorganisms. They revealed interactive effects of soil type and water deficit condition. The approach allowed us to identify key microbial taxa promoting drought adaptation and improve the understanding of drought effects on plant-microbe interactions.Plant-microbe interactions under drought stress are a popular research direction due to the widespread problem of the water crisis  amendments to paddy soils to reduce Cd bioavailability. The increased pH and phosphorus (P) in soils contributed to the decreased bioavailability of Cd and increased bacterial biodiversity. Also, Yu X. et al. found that using a Cd-immobilizing bacterial agent together with fermented organic matters in Cd-polluted soils could reduce plant (Houttuynia cordata) Cd uptake. Such amendments increased soil bacterial diversity.Soil heavy metal pollution is widespread  pollution is also another classic and severe environmental problem  is another heavy metal of concern   Bever,  to guideSzymańska et al. found that the isolated salt-tolerant endophytes could promote the growth of all tested plant species, suggesting a universal ability of endophytes to assist plant salt tolerance.Soil salinity is one of the most important abiotic factors limiting plant productivity . The authors revealed that the bacterial community was sensitive to environmental factors while the fungal community was more responsive to farmer intervention.Since the soil microbial community affects the growth, quality, and yield of plants, understanding the microbial ecology of the agroecosystem could provide hints for sustainable agriculture. Mai et al. found that different mangrove species harbor distinct microbial taxa in the sediments. Specific microbial taxa associated with the species R. apiculata contributed the highest functional activities related to carbon metabolism, carbon fixation, and methane metabolism. It indicates that mangrove-microbe interaction is species-dependent regarding the carbon cycle.Among natural ecosystems, the mangrove ecosystems represent unique and essential habitats serving as sensitive areas for shoreline ecological functions , Guangdong Natural Science Fund for Distinguished Young Scholar (2021B1515020014), and the National Natural Science Foundation of China  are gratefully acknowledged.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


As oral administration of vaccines is the preferred route due to its high patient compliance and ability to stimulate both cellular and humoral immune responses, it is also associated with several challenges that include denaturation of vaccine components in the acidic environment of the stomach, degradation from proteolytic enzymes, and poor absorption through the intestinal membrane. To achieve effective delivery of such biomolecules, there is a need to investigate novel strategies of formulation development that can overcome the barriers associated with conventional vaccine delivery systems. Nanoparticles are advanced drug delivery carriers that provide target-oriented delivery by encapsulating vaccine components within them, thus making them stable against unfavorable conditions. This review provides a detailed overview of the different types of nanocarriers and various approaches that can enhance oral vaccine delivery. The year 2020 was a devastating one for the healthcare systems of the world. A previously unknown variant of a known virus spread rapidly worldwide and quickly became one of its leading killers ,2. The bIf we focus on the traditional definition of vaccines only, they are always on a mission to either boost immunity against pathogens or to cut off the paths initiated by them. They are biological preparations that can provide active acquired immunity against particular infectious diseases through antigen recognition procedures . The proLow patient compliance related to invasive, inconvenient, or unpleasant routes using injections for vaccination has been a major factor in vaccine hesitancy for a long time. Hence, other routes for vaccine administration to raise patient compliance in both primary immunizations and subsequent boosts are being increasingly investigated ,10. The Nevertheless, there are several approaches that have been explored to deliver biopharmaceutical agents through the oral route. As discussed earlier, the oral route is not amenable to the delivery of many proteins and peptides, and advancements in drug delivery systems have changed the situation for vaccine delivery in many cases, including mRNA vaccine technologies . The selThe most preferred and well-liked mode of administration is oral delivery. Despite this, due to the inherent challenges posed by the digestive system, only a tiny portion of presently approved vaccines are oral formulations. Each portion of the GI tract offers a challenge with a different form of engineering that needs to be carefully handled to create an effective vaccination design . Most paAdvances in protein chemistry and biology permit the expression of peptides and proteins in eukaryotic cells of mammals, plants, animals, and fungi, as well as prokaryotic bacteria. Among all, yeast/single-celled fungi represent a model of choice for the expression of medically important proteins.Saccharomyces cerevisiae, Kluyveromyces lactis, and Pichia pastoris have the highest efficacy of heterologous gene expression.Among yeast and single-celled fungi, Despite being nonpathogenic, yeast cells used as vectors of vaccines can elicit an immune response in mammals and can be taken up by macrophages and dendritic cells .As we discussed yeast as a vector for vaccination, another important vector that can be used for vaccines is adenovirus. Adenoviruses, in general, do not cause considerable damage to human health. They have been used as a study model for eukaryotic cells, e.g., RNA splicing. Their molecular biology has been extensively studied, which has made them an excellent choice to become gene vector antigens. Since they can induce T-cell and B-cell immunologic responses to transgenes, they are suitable candidates for vaccine vectors. Their safety as vaccine carriers is established, as the risk of reversion to virulence is very slim in attenuated pathogens .Improved patient compliance:Numerous academic and biopharmaceutical researchers are working to create an alternative, needle-free oral vaccination technology in response to the drawbacks of injectable vaccines . Oral vaOral vaccine delivery is often simpler and less unpleasant, which may increase the probability that patients would request vaccination and return for follow-up shots .Capacity for mass immunization Self-administration: Due to their simplicity of administration and ability for self-administration, oral vaccinations have the potential to increase distribution compared to conventional injections. Self-administration reduces the requirement for qualified healthcare staff, making it perfect for extensive and quick vaccinations. Since the cost of launching a new vaccine can account for up to 25% of the cost, this might further cut the cost of vaccination programs .Simplified production and storage Low production cost: The gut, which is already populated with commensal bacteria, enzymes, nutrients, and lumens, will be exposed to oral vaccination. Therefore, unlike their injectable counterparts, oral vaccines do not need a high level of purity from a scaled-up and manufacturing viewpoint. However, extremely pure vaccines that are endotoxin-free must be used for injectable vaccines .No needle-associated risks: Each year, 5% of healthcare professionals experience needle-related danger, putting them at risk for blood-borne infectious diseases, including HIV/AIDS and hepatitis .Both IgG- and IgA-specific response: Antigen-specific mucosal secretory IgA antibodies and antigen-specific systemic IgG antibodies can be produced by vaccinations given by mucosal routes at all mucosal locations, not only the site of delivery .Nevertheless, despite being the most popular delivery method, oral administration has many challenges when reaching immune cells . There aAn oral vaccination must first be exposed to a very acidic pH, proteolytic enzymes, and bile salts, which will cause it to degrade in the digestive system (GIT) .For successful absorption and penetration across the intestinal walls, vaccinations must pass a variety of biological obstacles  in the intestinal lumen .Vaccines taken orally need to be absorbed and delivered to the right immune system cells. Typically, this is inefficient and requires the administration of high doses of vaccines ,24.Additionally, the brief antigenic exposure period to mucosal tissues contributes to the decreased absorption of antigenic particles. As a result, compared to their systemic equivalents, oral vaccinations may need multiple and larger doses to have a powerful and long-lasting immunogenic impact .However, oral administration of larger and repetitive oral doses of antigens might induce systemic nonresponsiveness , in which the antigens can be recognized as food or normal flora instead of eliciting protective immune responses ,26.Another significant obstacle to the development of oral vaccinations is the scarcity of powerful immunostimulants or mucosal adjuvants with minimal toxicity .A significant barrier to oral vaccination is the difficulty in measuring the real intensity of the immune response, particularly IgA, at different mucosal sites following oral administration .A short vaccination half-life might result from enzymatic breakdown, among other issues. Therefore, the scientific challenge is to significantly increase oral vaccination absorption from the conventional 1% .Different options for the oral delivery of vaccines are present nowadays, as shown in Commensal bacteria and food antigens are both relatively abundant in the gut environment. Intestinal epithelial cells (IECs) that were formerly thought to just serve as a physical barrier to the outside world are now known to play a more important role in the induction of innate and adaptive immunity and in the maintenance of immunological homeostasis. IECs are the pioneer cells encountering intestinal pathogens, and they can work as immunological sensors detecting pathogens through different classes of pattern recognition receptors (PRRs), including nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and Toll-like receptors (TLRs) .Ligands such as wheat germ agglutinin (WGA) that target N-acetyl-D glucosamine and sialic acid residues expressed by enterocytes throughout the GI tract have been well explored for oral drug delivery . The gly® in a mice model [A study conducted by Davitt et al. concluded that α-GalCer strongly enhanced antigen-specific antibody responses following oral coadministration with Dukoralce model .M cells might be effective delivery vehicles for oral vaccines . The folNanotechnology holds great promise in the oral delivery of vaccines by incorporating them within the nanoparticulate carrier, which not only improves their stability against proteases but also helps in rapid absorption through the gastrointestinal tract (GIT) . An ideaThe current review will provide a description of the different nanocarriers that can be used for oral vaccine delivery, as shown in i.Polymeric NPs;ii.Lipid-based systems;iii.Inorganic NPs;iv.Niosomes;v.Advanced vesicular drug delivery systems.Transferosomes and ethosomes also come under the class of advanced vesicular drug delivery systems, but these systems are not discussed in detail in this review, as transferosomes and ethosomes majorly show applications in transdermal drug delivery and have very limited application in the oral delivery of drugs .These are small-sized particulate carriers that can encapsulate, adsorb or conjugate any antigen into their core or on the outer surface . Over a ➢Mucoadhesive NPs;➢Stimuli-responsive NPs;➢Specific ligand-bearing NPs.For targeted delivery, a wide range of polymers with the required physical and chemical properties can be used . AdvanceSalmonella typhi porin proteins into PLGA. This encapsulation showed enhanced efficacy due to the protection of antigens from degradation in the stomach pH after delivery through the oral route. pH-sensitive polymers protect antigens from the harsh gastric environment and release antigens only in the intestine region [Synthetic polymers such as polyesters, i.e., poly lactic acid (PLA) and poly lactic-co-glycolic acid (PLGA), have been most commonly used to prepare biodegradable nanoparticle-based vaccines that provide prolonged antigen release with a single-dose administration . On the e region . Zhu et e region . Researce region . By conte region . Crosslie region .Another class of synthetic polymers is polyanhydrides. They are also biodegradable but less acidic than polyesters and help to enhance the stability of entrapped molecules. These polymers enhance immunogenicity without the need for additional adjuvants. Mannan-coated methacrylic acid-based copolymer is also an example of this type of synthetic polymer, which provides a pH-dependent release of entrapped antigens along with the activation of activated phagocyte cells (APCs) . The useA literature survey explained the application of naturally obtained polymers, e.g., chitosan and its derivatives, in the oral delivery of proteins and peptides against ovarian and breast cancer . Chitosa➢LiposomesLiposomes have gained immense importance in drug delivery due to their ability to encapsulate both hydrophobic and hydrophilic components. These are composed of a self-assembly of phospholipids constituting an inner aqueous core and an outer lipid bilayer . HydrophLiposomes exhibit many advantages, including particle uptake by M cells and the ability to induce cytotoxic T lymphocytes by targeting antigens to endogenous processing pathways . Modific➢NanoemulsionThis is an isotropic system formed by adding two immiscible liquids, i.e., oil and water, and is stabilized by using a suitable quantity of surface-active agents to obtain nanosized droplets. Usually, they have a size range of 20 to 200 nm . Size mi➢Immunostimulating complexes (ISCOMs)In 1984, ISCOMs were first studied as carriers for vaccine delivery. They are defined as self-assembled complexes that are spherical in shape. Usually, they have a particle size of 40 nm. Their composition includes antigens, cholesterol, phospholipids, and Quil A saponins . ISCOMs Inorganic NPs have emerged as novel carriers for the oral delivery of vaccines because of their interesting physicochemical features . ModificNiosomes are nonionic surfactant vesicles having a size range on a nanometric scale. Niosomes are prepared from a mixture of nonionic surfactants of the alkyl or dialkylpolyglycerol ether class and cholesterol followed by hydration in aqueous media. They serve as novel drug delivery carriers for drugs and are gaining importance in the oral delivery of vaccines . The del➢ExosomesExosomes, which are spherical vesicular structures derived from endosomes and range in size from 40 to 160 nm in diameter, are composed of several kinds of proteins and lipid bilayers. They are abundantly present in all physiological fluids and tissues and are released by various cell types . ExosomeExosomes can be either naturally occurring or artificially produced. Animal- and plant-origin exosomes make up the second division of natural exosomes. Exosomes originating from animals are further split into normal exosomes and exosomes derived from tumors. All normal cell types, including T cells, beta cells, macrophages, dendritic cells, and mesenchymal stem cells, can release exosomes. For instance, mesenchymal stem cells can differentiate into multiple adult cell types. MSCs can readily adapt to the tumor microenvironment and have a high exosome production rate. Normal exosomes can be found in bile, breast milk, blood plasma, urine, and saliva . The abiTo deliver the COVID-19 vaccine, mRNA-based exosomes have recently been identified. Tsai et al. precisely fabricated the LSNME-SW1 vaccine, which generated wide immunities against COVID-19, and confirmed the utilization of exosomes for delivering the required mRNA into the host cell in vivo and in vitro .➢ColloidosomesThe self-assembly of colloidal particles forms microcapsular structures known as colloidosomes. Such particles have a hold over the mechanical features of colloidosomes, which includes size, permeability, and strength . Based o➢AquasomesAquasomes are self-organized nanoparticulate carrier systems composed of three basic layers. Among these layers, the first one is the innermost called the core, the second layer is a middle coating of carbohydrate material, and the third layer is of active pharmaceutical ingredient (API) moieties that are to be administered and are embedded in the carbohydrate layer . The cor➢PolymersomesAmphiphilic copolymer allows polymeric vesicles to self-assemble into polymersomes . Polymer➢PhytosomesAqueous-based phytomedicine–phospholipid complexes generate phytosomes, which are colloidal, small-sized vesicles, such as liposomes, having micellar or hexagonal nanostructures. They are sometimes referred to as pharmacosomes in the pharmaceutical industry ,108. Lim➢EmulsomesEmulsomes are a new type of vesicular system with an internal core consisting of solid fat and an outer phospholipid bilayer around it . Cholest➢EnzymosomesRecently, enzymosomes have been reported as innovative vesicles. Enzymosomes are synthesized by incorporating enzymes within carriers similar to living cells having an enriched lipid background . Enzymos➢SphingosomesComplex stability issues associated with liposomes lay the foundation of sphingosomes’ formation. The instability of liposomes occurs due to the hydrolysis of the ester bond of phospholipids. This hydrolysis can be prevented using lipids having ether or amide linkage in place of an ester linkage. As a result, sphingolipids are currently employed to create stable a form of liposomes named sphingosomes  and are The use of NP-based delivery systems makes it possible to administer vaccines orally, which is the preferred route due to its high patient compliance rate, capability for mass immunization, ease of use for less-trained staff, and lack of risk from needle-associated injuries. Prior to this, no vaccine has been delivered through the oral route, excluding live-attenuated or inactivated microorganism-based vaccines using nanoparticles. NPs enhance immunogenicity and provide stability and protection to antigens against the unsuitable gastric environment. The strong immune response of NPs could be by virtue of their nanosize that mimics the size of pathogens. Apart from size, other features, such as physicochemical characteristics, also influence the immune response of NPs. The stability of antigens in the GIT has been significantly improved by several mucoadhesive and stimuli-responsive biodegradable polymers, including PLGA, PGA, chitosan, and alginate alone or in combination. Liposomes and other vesicular delivery systems can incorporate both hydrophilic and hydrophobic components within them, and they can also be fabricated by covering them with different polymers to increase their stability against denaturation. Ligand-specific targeting to M cells, epithelial dendritic cells, and enterocytes can overcome poor permeation of antigens through the mucosal barrier of the intestine associated with oral delivery. It can be proposed that the hindrances accompanied by the GIT can be prevented by using nanotechnology that not only provides selective targeting to intestinal immune cells but also reduces toxic side effects.Nanoparticle-based oral vaccines can be developed by selecting a suitable carrier into which vaccines can be incorporated that can provoke a better immune response. The current review suggests that vaccine delivery through the oral route has many more benefits as compared to the parenteral route. In addition to these advantages, the oral route for vaccine delivery also encounters certain challenges, which include the degradation of antigens in unfavorable conditions. Nanotechnology provides us with carriers that can overcome the limitations associated with the oral delivery of vaccines. The only remaining question is whether they can be made using economically viable methods to enable mass vaccination at a cheap cost, as oral immunization has demonstrated, which is especially useful in impoverished countries.


Resistant chronic migraine is a highly disabling condition which is very difficult to treat. The majority of the treatments for migraine prophylaxis are nonspecific and present weak safety profiles, leading to low adherence and discontinuation. Currently, monoclonal antibodies (mAb) targeting the trigeminal sensory neuropeptide, calcitonin gene-related peptide (CGRP), are available for migraine prophylaxis being the first drugs developed specifically to target migraine pathogenesis. The main objective of the current work is to carry out a systematic review of randomised controlled trials that specifically analyse the effectivity and safety of anti-CGRP mAb, comparatively to placebo, in patients with resistant chronic migraine and possibly fill the literature gap or be a source of information to health professionals. Additionally the current knowledge on migraine, particularly resistant chronic migraine, was revisited and summarised.Literature search was carried out on MEDLINE, Scopus, Science Direct and ClinicalTrials.gov database, from inception to December 2021. Articles were selected according to prespecified criteria of inclusion and exclusion. Efficacy and safety outcomes included were: change from baseline in monthly migraine days (MMD); ≥50% reduction of MMD values from baseline; change from baseline in monthly acute migraine-specific medication days (MAMD); Migraine-specific Quality of Life Questionnaire (MSQ); and registered adverse events. Additionally, we used the Cochrane risk of bias tool (RoB 2) to assess the risk of bias of the included studies.Four studies were included in this systematic review, involving 2811 resistant chronic migraine patients, 667 in a study using erenumab, 838 in a study using fremanezumab and 1306 in two studies using galcanezumab. When compared to placebo, all investigated anti-CGRP mAb and respective doses demonstrate effectiveness in decreasing MMD, reducing acute medication use and improving the MSQ scores, including, sometimes, reversion of chronic to episodic migraine (efficacy outcomes). Regarding the safety outcomes, the number and type of adverse events did not differ between anti-CGRP mAb-treated and placebo groups.Anti-CGRP or anti-CGRP receptor monoclonal antibodies are a promising preventive migraine therapy which can be particularly useful for resistant chronic migraine patients. Migraine is a chronic neurological disorder with paroxysmal features and episodic manifestations characterised by multiphase attacks of head pain associated with other symptoms of neurologic dysfunction, such as sensitivity to movement, photo- and phono-phobia, nausea and vomiting. A migraine attack has three phases: premonitory (prodrome), headache phase and postdrome; each has distinct and sometimes disabling symptoms. About 20–25% of migraine patients have a fourth phase called aura . MigrainAccording to the Global Burden of Disease Study 2016, migraine is the second leading cause of disability and carries significant personal, social and economic burdens . MigrainMigraine presents itself as three major types, according to the International Classification of Headache Disorders-3 (ICHD-3): migraine with aura, without aura and chronic migraine . MigrainAlthough the pathophysiology of migraine is still, at this date, not completely understood, several recent exhaustive reviews have gathered and explained the multifactorial causes of this neurologic condition –80. Suchnucleus caudalis in the brainstem, and from there direct to the periaqueductal grey matter, sensory thalamic nuclei and somatosensory cortex . No language restriction or other limits were used.After screening of studies, duplicates were removed, and the remaining studies underwent a two-stage screening process. The first stage involved title and abstract screening. The second stage involved conducting full-text reading to exclude irrelevant trials. Furthermore, reference lists of included studies were screened to consider additional relevant studies. Two authors independently screened the studies.versus placebo of patients reaching ≥ 50% reduction in MMD from baseline over 3 months and (iv) change in MSQ scores. Safety outcomes included the presence of any type of adverse effect and their grade.The following three categories of data were extracted from the included studies: (a) baseline characteristics, (b) efficacy and (c) safety outcomes. Baseline characteristics of the studies included: (i) name of the first author; (ii) year of publication; (iii) national clinical trial (NCT) identifier; (iv) number of previous treatment failure sample size; (v) mean age of participants; vi) percentage of females; (vii) MMD at baseline; (viii) MAMD at baseline; (ix) mean time since initial migraine diagnosis (years) and x) MSQ scores. Efficacy outcomes included (i) change in MMD; (ii) variation in MAMD; (iii) proportion and odds ratio The Cochrane Collaboration’s risk of bias tool (RoB 2) was used to assess the risk of bias of the included randomised placebo-controlled trials . This riLiterature search yielded a total of 105 studies. Six duplicate studies were removed before screening. After screening, of the remaining 99 studies, 90 were excluded as they did not meet the inclusion criteria. Full-text screening of the remaining 9 studies resulted in a further elimination of 5 studies that did not match all inclusion criteria or had any exclusion criteria. Finally, only 4 articles were included in the qualitative synthesis  are summarised in Table versus placebo groups. The chances of a patient in the intervention groups to have a 50% or great response, are higher than those of patients in the placebo groups, as confirmed by the calculated odds ratio (OR) for each study. Concerning the patients’ quality of life and disability, a significant improvement was also verified from baseline values in the MSQ scores in anti-CGRP mAb-treated patients versus placebo treated patients .TablThe safety outcomes measured in the studies are summarised in Table The four RCTs included in this synthesis were scored as presenting “low risk” of bias for the following domains of the Cochrane risk of bias tool RoB 2): randomisation process, deviations from the intended interventions, missing outcome data and selection of the reported results  analyseThe baseline characteristics of the four studies included in this review are comparable between studies and across all treatment groups, with similar mean age, MMD, MAMD, years since initial migraine diagnosis and MSQ scores Table . These fMoreover, the safety outcomes including main adverse events, serious adverse events and adverse events that led to treatment discontinuation are also in line with previously published results –42. ThesTo the best of our knowledge, this is the first study to collect and discuss data about the efficacy and safety of anti-CGRP mAb in the subgroup of resistant chronic migraine patients, with previous failures to several preventive medications. The decision to include only randomised placebo-controlled clinical trials was made to guarantee high-quality evidence. Moreover, the analysis included subgroup evaluation corresponding to the most used anti-CGRP mAb doses for each molecule to preserve consistency about drug dosing.The current review presents, however, several limitations. The studies excluded patients with serious or unstable medical conditions such as major cardiovascular conditions, which can limit the extrapolation of the safety results to all patients with chronic resistant migraine. However, treatments with anti-CGRP mAb have shown to be safe in patients with previous cardiovascular risk factors and have not shown significant differences in adverse outcomes, cardiovascular or others, when drug treatment and placebo groups were compared . MoreoveAlthough RCTs remain the most rigorous scientific method for evaluating the effectiveness of health care interventions, real-world studies  have grown increasingly relevant in the scientific world in recent years . DespiteFurther studies with larger numbers of patients, adequate time periods to measure differences in long-term complications or adverse events and real‐world data would be required, in the future, to confirm the effectiveness, tolerability and safety for longer periods of treatment and in populations with more comorbidities. Furthermore, a survey for physicians involved in migraine care, carried out by the European Headache Federation with the endorsement of the European Migraine & Headache Alliance, demonstrated the necessity of more evidence regarding the management of these patients as well as clearer guidelines for physicians . Other fMigraine is associated with high disability and, despite being a prevalent disorder, is often underdiagnosed . It is cAnti-CGRP mAb appear to have a great potential for the treatment and reversibility of resistant chronic migraine. In this review, resistant chronic migraine patients showed better ≥ 50% response rates, lower number of days with migraine episodes, lower acute migraine-specific medication use and improved scores of patients reported quality of life. Moreover, in the population of patients with resistant chronic migraine studied in this review, monoclonal antibodies raised against CGRP or CGRP receptors present adverse events and tolerability profiles similar to those of placebo. These positive results obtained in a quite difficult to treat group of patients, who have not benefited from or tolerated several previous standard-of-care treatments, appear significant and exciting. However, longer treatment periods could uncover currently unknown risks. The physiological changes that end an attack and the factors that influence remission or progression to persistent symptoms in migraine over time, are questions that remain unanswered. Therefore, there is the need to carry out additional RCTs with larger populations and real‐world data studies, in a near future, in order to answer these questions and guide the development of disease-modification strategies and determine the importance of these innovative treatments for migraine prevention.


Tendons are collagen-rich musculoskeletal tissues that possess the mechanical strength needed to transfer forces between muscles and bones. The mechanical development and function of tendons are impacted by collagen crosslinks. However, there is a limited understanding of how collagen crosslinking is regulated in tendon during development and aging. Therefore, the objective of the present review was to highlight potential regulators of enzymatic and non-enzymatic collagen crosslinking and how they impact tendon function. The main collagen crosslinking enzymes include lysyl oxidase (LOX) and the lysyl oxidase-like isoforms (LOXL), whereas non-enzymatic crosslinking is mainly mediated by the formation of advanced glycation end products (AGEs). Regulators of the LOX and LOXL enzymes may include mechanical stimuli, mechanotransducive cell signaling pathways, sex hormones, transforming growth factor (TGF)β family, hypoxia, and interactions with intracellular or extracellular proteins. AGE accumulation in tendon is due to diabetic conditions and aging, and can be mediated by diet and mechanical stimuli. The formation of these enzymatic and non-enzymatic collagen crosslinks plays a major role in tendon biomechanics and in the mechanisms of force transfer. A more complete understanding of how enzymatic and non-enzymatic collagen crosslinking is regulated in tendon will better inform tissue engineering and regenerative therapies aimed at restoring the mechanical function of damaged tendons. Tendon is predominantly composed of collagen, which is hierarchically arranged in fibrils, fibers, and fascicles . The strThe interfibrillar and intrafibrillar crosslinks between collagen molecules are formed by both enzymatic and non-enzymatic processes, which contribute in a unique way to tendon functions and changes over time. Collagen crosslinking that occurs during embryonic tendon development appears to be mainly regulated by enzymatic processes, such as by LOX activity . In collNon-enzymatic crosslinks, such as AGEs (with glucosepane being a predominant AGE), are spontaneously formed through glycation processes and yield divalent crosslinks between neighboring fibril residues  and failure mechanisms . However, future work will need to directly compare how enzymatic vs. non-enzymatic crosslinks impact tendon mechanical properties. Despite the important and complex role collagen crosslinking plays in tendon formation and pathology, it is less clear how these crosslinks are regulated. A thorough understanding of the regulation of enzymatic and non-enzymatic crosslinks is needed to better determine how tendon mechanical properties are regulated, to guide the development of mechanically functional engineered tendon tissues, and to better characterize tendon pathogenesis. Further, manipulation of crosslinks may be applied to a variety of tissue engineering techniques that use collagen-based biomaterials to modulate mechanical properties and failure mechanisms. Therefore, the present review aimed to highlight recent findings on the potential regulators of enzymatic and non-enzymatic collagen crosslinks in tendon, as well as to discuss the limited knowledge on collagen crosslinking in tendon development, disease, and aging. To further develop novel effective treatments for damaged tendons, an improved understanding of collagen crosslinks and the mechanisms governing their formation is needed.The effects of enzymatic and non-enzymatic collagen crosslinks on tendon mechanical properties have been explored in several recent reviews  via the cFollowing secretion and hydroxylation of the triple-helical procollagen, LOX catalyzes the formation of Hyl and lysine residues into reactive aldehyde species. In turn, these species spontaneously link to aldehydes on adjacent collagen molecules by covalent bonds to form immature divalent crosslinks . Immaturi.e., porosity) between the tropocollagen molecules within a collagen fibril to be around 2 nm , thereby inhibiting LOX diffusion. Furthermore, the molecular weight of LOX (32 kDa) approaches the upper limits of collagen type I pore size, estimated to be between 5.7 kDa and 48 kDa, which may limit LOX ability to diffuse into the fibril  are driven by LH1 and LH2 during fibrillogenesis (A recent study has suggested that these LOX-mediated crosslinks are formed at the fibril surface during fibrillogenesis because the LOX molecule (3.7 nm radius) may be too large to diffuse within the collagen fibril  and a higher crosslink density , raise intriguing questions regarding the specific role of enzymatic crosslinks in these tissues and how they are differentially regulated.The different crosslinks and concentrations within various tendon types, particularly tendons of different functions (etc.), particularly within critical transition regions of the enthesis. A better understanding of these phenomena may aid in the development of tissue-engineered constructs and regenerative therapies targeted at treating evulsion injuries.The mechanical properties and structure of tendon change spatially as the tissue transitions from the tendon itself (mainly collagen) to its insertion site into the bone . The tendon-bone interface (enthesis) is particularly pertinent because partial- and full-thickness tendon tears at the enthesis are a primary cause of rotator cuff injury  and folding of the C-terminal  to form three or more disulfide bonds  interaction with the copper-dependent cofactor ; ii) interaction with the LTQ prosthetic group at the LOX active site  . BAPN treatment resulted in a mechanically weaker construct  and abnormal collagen fibrils that resembled Ehlers-Danlos collagen phenotypes  changes throughout tendon development, demonstrating greater structural order  in mature compared to fetal tendons  profile (through treatment with 4-aminopyridine) show an increased calcaneal tendon elastic modulus relative to controls, without changes in LOX expression. However, chick embryos treated with rigid and flaccid paralysis (unloading) phenotypes (through treatment with decamethonium bromide or pancuronium bromide) have a decreased calcaneal tendon elastic modulus that corresponds to decreased LOX activity. Interestingly, a hypermotility plus BAPN treatment does not significantly alter the elastic modulus  favors ECM degradation and tissue remodeling . Excessive loading increased collagen maturation as well as LOX and LH2 positive cells. Excessive loading plus BAPN treatment suppressed the relative increases in LOX and LH2 positive cells, as well as collagen maturation  and stabilizing  proteins.hPDL cells embedded within a collagen gel respond uniquely to different magnitudes of mechanical loading. Low tensile strain magnitudes (3 %) upregulate versus energy-storing tendons   but Young’s modulus and HP crosslinks are not impacted, relative to controls. Together, this suggests that hypoxia may mediate LOX expression and activity to impact tissue modulus. Hypoxia and BAPN treatments were also applied to bovine ACL, PCL, patellar tendon, and knee meniscus explants; the same trends in modulus were detected, although LOX expression was not assessed in these tissues (The oxygen tension of tendon  oxygen)  or bone  culture . When BALOX via the HIF-1 pathway, specifically, through the hypoxia-response element located in the promoter region of LOX  induces activation of While LOX appears to be impacted by hypoxia, the specific cellular pathways are poorly understood, particularly in tendon. Further investigation of this topic has potential to significantly impact the understanding of the hypoxia-dependent mechanisms governing enzymatic crosslinking in tendon.LOX and LOXL1-4 expression in human trabecular meshwork  cells via both Smad and non-Smad pathways (MAPK/JNK and AP-1) through TGFβR1 and TGFβR2  have altered tendons  that exhibit increased LOX-induced mature crosslinks within collagen type I C-telopeptide domains, but LOX itself does not appear to be affected through post-translational modifications .e.g., the KGHR motif) and collagen crosslinking. The dual binding of these glycoproteins to fibrillar surface domains demonstrates that LOX-mediated crosslinking is modulated through a variety of interactions that are not yet well understood in tendon.Together, these studies on TSP-1 and fibromodulin highlight the importance of further investigating the interactions between glycoproteins that bind to the surface of collagen fibrils (One study on CypB (an ER-resident chaperone protein)-null mice, used as a model for studying recessive osteogenesis imperfecta, showed that tail tendons exhibit poorly organized and abnormal morphology. CypB contributes, but does not appear to be essential, to the P3H complex that regulates LH isoforms and procollagen crosslinking  have reduced mechanical properties  and LOX activity, but no changes in collagen content. Interestingly, LOX activity is more severely inhibited with estrogen treatment than LOX mRNA, suggesting estrogen may affect LOX primarily post-translationally  and tendons  and rates of injury may be attributed to the different mechanical roles of the tissues, but also several other confounding factors . Together, these results suggest LOX may be partially controlled post-translationally by estrogen and justifies further investigation into the effects of estrogen on collagen crosslinking  within tendon.When comparing the results of these studies, it is important to note that while Achilles tendons in females appear to be more protected from injury than males, females have an increased risk of ACL rupture, especially during menstruation , such as tendon, has the potential to facilitate restoration of pre-injury mechanical function. Similarly, injection of BAPN combined with controlled exercise has been used to improve fiber alignment and reduce the risk of reinjury in adult horse tendons , appeared to be less fibrogenic compared to TGFβ1-only treated controls  and young (ages 27 ± 2 years) human patellar tendons found aged tendons have decreased total collagen level, increased levels of both HP and LP enzymatic crosslinks, and increased pentosidine level (as a representative AGE), compared to young tendons  increasei.e., fiber sliding, helical rotation, etc.). One study explored the age-specific response of equine SDFTs, an energy-storing tendon, and found that aged tendons have a decreased ability to withstand fatigue loading (cyclic loading) due to aged fascicles being less capable of helical rotations , cellular (decreased cell proliferation and stem/progenitor cell quantity), and mechanical properties (reduced modulus and strength), beyond just alterations to AGE-mediated crosslinks, occur in tendon during the aging process and have been recently reviewed in detail , a marker of advanced glycation, and increases the tendon solubility of diabetic mice, suggesting hyperglycemic-induced glycation and AGEs can be therapeutically targeted.The slow turnover rate of collagen molecules within tendon makes it a tissue particularly susceptible to excess free glucose associated with hyperglycemia, as seen in diabetic patients, and it may induce excessive AGE accumulation at more dramatic rates than healthy counterparts . DiabetiAGE accumulation is also affected by the diet. Mice fed a normal diet with a large AGE content show higher levels of AGEs  in both Achilles and tail tendons (with the Achilles having higher levels than the tail tendons) compared to mice fed a high-fat low-AGE content diet  in tendon.Ribose and MGO are commonly used treatments that induce AGE formation and can be used to study the effects of non-enzymatic crosslinking on tendon. These treatments are effective at accelerating and simulating AGE accumulation starting from glucose in vivo  and pentosidine (as a representative AGE), but decreased total collagen content . In anotin vivo glucose reactions. Although, similarly to ribose, excessive MGO treatments can be less physiologically relevant , differently from untreated control fibrils that exhibit strain-stiffening and strain-softening . Notably, these findings contradict a similar work on rat tail tendons using MGO-derived crosslinks that suggests AGE accumulation impacts tissue mechanics through fiber-level mechanisms, not alterations to fibrils  and mechanical testing platforms. However, it will be important to identify how different crosslinking mechanisms affect fibril and fiber-level mechanics in tendon.Glutaraldehyde is another potent inducer of non-enzymatic crosslinks. i.e., glucosepane has a much larger and obstructive structure than DOGDIC) altering its binding characteristics. Experimentally derived observations to validate these models will be an important future direction as well as identifying the specific binding locations. Overall, these findings justify both further investigation of various AGEs in tendon and the development of new methods to study AGEs in vivo.The effects of glucosepane, the most abundant lysine-arginine-derived AGE, have been recently investigated using computational modeling. i.e., being more brittle), demonstrating that dynamic crosslinking and glycation  are necessary for proper tissue function  are needed for proper collagen crosslinking both during and after fibrillogenesis, which mediates the mechanical formation of tendon and may distinguish functionally distinct tendons . However, the mechanisms governing enzymatic crosslinking are not well understood and require further investigation. Potential regulators of enzyme production at the cellular level may include mechanical stimuli, mechanotransducive cell signaling pathways, sex hormones, TGFβ family, hypoxia, and interactions with intracellular or extracellular proteins.Enzymatic and non-enzymatic collagen crosslinking plays a role in regulating the mechanical function of tendon. Multiple collagen crosslinking enzymes (AGE crosslinks derived from non-enzymatic interactions resulting from free glucose impact tendon function. AGEs, which accumulate during the normal aging process, can be accelerated under diabetic conditions and may also be affected by diet. The specific consequences of AGE accumulation are not entirely understood but may alter both fiber and fibril behavior. An improved understanding of the exact location and process through which these crosslinks form is needed. Overall, enzymatic and non-enzymatic crosslinks affect tendon biomechanics and, thus, a better understanding of their regulators can advance future clinical treatments for tendon disease, injury, and age-related conditions.