Aviation and space medicine face many common musculoskeletal challenges that manifest in crew of rotary-wing aircraft (RWA), high-performance jet aircraft (HPJA), and spacecraft. Furthermore, many astronauts are former pilots of RWA or HPJA. Flight crew are exposed to recurrent musculoskeletal risk relating to the extreme environments in which they operate, including high-gravitational force equivalents (g-forces), altered gravitational vectors, vibratory loading, and interaction with equipment. Several countermeasures have been implemented or are currently under development to reduce the magnitude and frequency of these injuries. Cervical and lumbar spine, as well as extremity injuries, are common to aviators and astronauts, and occur in training and operational environments. Stress on the spinal column secondary to gravitational loading and unloading, ± vibration are implicated in the development of pain syndromes and intervertebral disk pathology. While necessary for operation in extreme environments, crew-support equipment can contribute to musculoskeletal strain or trauma. Crew-focused injury prevention measures such as stretching, exercise, and conditioning programs have demonstrated the potential to prevent pre-flight, in-flight, and post-flight injuries. Equipment countermeasures, especially those addressing helmet mass and center of gravity and spacesuit ergonomics, are also key in injury prevention. Furthermore, behavioral and training interventions are required to ensure that crew are prepared to safely operate when faced with these exposures. The common operational exposures and risk factors between RWA and HPJA pilots and astronauts lend themselves to collaborative studies to develop and improve countermeasures. Countermeasures require time and resources, and careful consideration is warranted to ensure that crew have access to equipment and expertise necessary to implement them. Further investigation is required to demonstrate long-term success of these interventions and inform flight surgeon decision-making about individualized treatment. Lessons learned from each population must be applied to the others to mitigate adverse effects on crew health and well-being and mission readiness. The crew of high-performance jet aircraft (HPJA), rotary-wing aircraft (RWA), and spacecraft are subjected to extreme occupational conditions that elevate their risk of pre-flight, in-flight, and post-flight musculoskeletal pathology and disability. In astronauts, musculoskeletal issues are the second most common cause of in-flight complaints (second only to sleep disturbance) with an incidence of 3.34 events/person-year . MusculoThe risks are compounded in some astronauts who were former HPJA or RWA pilots and later participate in spaceflight. In one study of intervertebral disk (IVD) injury in a group of 321 astronauts, over half (178) of the astronauts were former HPJA pilots . MoreoveMusculoskeletal pain and injury in these populations have been attributed to several aspects of flight including: microgravity; high-g-forces acting parallel to axis of the spine high-Gz, ; vibratoTo counter the debilitating effects of the occupational injuries associated with the aerospace environment, many efforts have been made to develop both aircraft- and pilot-focused countermeasures. Many of the countermeasures developed out of needs identified in HPJA crew, but application to astronauts and crew of other aircraft should be considered due to their similarly extreme exposure. This study aims to review the countermeasures available to aerospace medicine professionals in support of their mission to protect crew from the conditions that might otherwise predispose them to injury and disease. Some potential countermeasures include exercise, stretching, traction, behavioral interventions, re-design of cockpit or equipment, helmet re-configuration and counterweights, and anti-vibration seating .In order to develop effective countermeasures, it is critical to understand the underlying mechanism of injury. Loss of gravitational loading in spaceflight results in decreased bone mineral density (BMD), change in IVD composition, flattening of the spinal curvature, muscle atrophy, and other myofascial changes in astronauts . BMD losAlthough studies of astronauts immediately post-flight detected no appreciable change in IVD height, analog studies suggest unloading of the spine allows increased water absorption by proteoglycans of the IVD, leading to an increase in IVD volume . IVD expIn microgravity, the vertebral body BMD changes and loss of paraspinal lean muscle mass predispose to herniated nucleus pulposus (HNP) , 4 John. The risBone mineral density loss can also predispose to fracture, but none of over 50 fractures in active United States astronauts can be definitely attributed to exposure to microgravity . Two astIn the astronaut population, HNP incidence is not increased with longer duration missions on the ISS, likely due to protocols that limit vertical positioning and ambulation within the first 24 h post-flight and reconditioning coaches to facilitate protected recovery for the first 2 weeks post-flight . The abiCountermeasures to preserve muscular strength and endurance and BMD in astronauts include aerobic and resistive exercise together with nutritional supplementation, high dose vitamin D, and bisphosphonates . A studyAnother consequence of microgravity is space adaptation back pain (SABP). SABP is a self-limited condition involving pain predominantly in the lumbar spine during the first several days of spaceflight . IncreasThe goals of countermeasures in spaceflight include minimizing adverse health outcomes and lifetime health risks, facilitating in-flight performance, and optimizing post-flight recovery . InjurieWhole body vibration is a risk most prominent in RWA crew but also present in HPJA crew and astronauts . WBV resHigh-Gz exposure has been established as a risk factor for neck pain based on the relative increased incidence of neck pain in pilots of high-Gz aircraft compared to pilots of intermediate- and low-Gz aircraft . High-GzEvidence for compression of IVD during flight is supported by decreased body height following aerial combat maneuvers . ChronicHigh-Gz loading in conjunction with HSM is further exacerbated with several head positions and movements, most notably the “check six,” a combat maneuver in which the pilot maximally rotates the head to look behind the aircraft . FurtherEfforts have been made to mitigate these environmental effects with helmet counterweights, anti-vibration seats, and re-configuration of cockpits and equipment . The neeFor HPJA crew, efforts to consider head position before initiating high-G maneuvers have shown to be protective against neck pain . In one Numerous countermeasures have been developed to mitigate the musculoskeletal effects on aircrew and astronauts including exercise, stretching, reconditioning, traction, and behavioral interventions . Even inMuch like the evidence in support of directed exercise interventions for non-specific neck pain in the general population, studies have also demonstrated success in RWA and HPJA pilots using a prescribed resistive exercise regimen with elastic bands to improve strength and endurance of cervical spine musculature , mitigatp < 0.05)  . Future Other novel interventions have been proposed including trampoline training which is suggested to provide low-intensity, repetitive muscular endurance exercise . In a stFor HPJA pilots, cervical vertebrae are especially vulnerable to injury during the sudden acceleration of ejection . Pilots Astronauts and aircrew may be at increased risk also because of their increased proclivity toward physical activity and the demands placed on them by rigorous training . In astrForced unnatural posture for long periods is another major contributor to musculoskeletal disability in pilots . Many HPChanges to cockpit equipment have also been considered. For instance, the angle of the seat affects the incidence of pain in pilots of the aircraft . The oriThe unique environmental conditions of flight together with the rigorous training and mission-related tasks conducted by aircrew and astronauts place them at risk for musculoskeletal injuries that have the potential to impact crew readiness and mission success. The astronaut population is small and the relative abundance of HPJA and RWA crew may assist in understanding the musculoskeletal complaints seen in astronauts. One must consider, based on similar type and magnitude of exposures, that if a particular problem manifests in one population, it may be a harbinger of problems to come in the other populations of crew. In this way, collaborative understanding of musculoskeletal issues will help medical providers and researchers to stay ahead of potential issues and develop countermeasures to ensure continued crew health and safety.The impending launch of commercial spacecraft could pave the way to increased access to space in which case a more diverse population of individuals will be subjected to the musculoskeletal risks of flight. Moreover, interest in long duration missions including those to establish lunar colonies and reach Mars, will result in prolonged exposure to the spaceflight environment beyond that which has been previously studied. These potential developments underscore the importance of understanding the musculoskeletal risks and developing appropriate countermeasures. Even beyond the air and space crew populations, many of these findings and countermeasures may have application to other populations. Neck and back pain affect a significant share of the general non-flying population and pose a similar problem in other occupations including military parachutists, infantry soldiers, and law enforcement largely as a result of eccentrically loaded HSM , as wellAs a result of the occupational hazards of the flight, crew are exposed to numerous risks that have the potential to impact crew health and readiness. Every effort must be made to address these risks and implement effective countermeasures. The risks and exposures are varied and there is evidence of both acute and chronic injuries in crew. The resulting distribution of pain across age groups is bimodal, with the largest share of those affected being either early career aviators being introduced to flight or those accumulating more hours late in their career . In the The goal of countermeasures is to prevent irreversible injury but, in some cases, when this is not possible, countermeasures must be employed to mitigate existing injury or rehabilitate patients. Some examples of countermeasures for mitigation of existing injury include pharmacological interventions such as anti-inflammatory medications, massage, acupuncture, heating pad, traction, physiotherapy, and chiropractic or osteopathic manipulation . HoweverNumerous approaches have been taken to develop countermeasures including those focused on aircraft design, crew strength and behavior, and protective equipment worn by crew. Each approach has its unique advantages and disadvantages. Aircraft design interventions, for example, are likely to require the most upfront cost but impose the least time and effort burden on crew. While exposures are similar between crew, interventions may need to be individually tailored to the airframe. For instance, seat interventions in HPJA require titration of seatback angle to optimize G-tolerance with lowered incidence of neck pain. In RWA, seat interventions require focus not only on pilot postural support but also on vibration reduction and crash-landing protection. Vibration reduction can be tackled not only through seating interventions, but also through improved engine and airframe design.Effective countermeasure development must be crew-friendly and take into account barriers to implementation. Time limitations imposed by busy flying schedules result in poor adherence to exercise countermeasures . Other bIt is important that countermeasures are made available to all crew who may be at times more vulnerable to injury than the pilots . BackseaIt is imperative to acknowledge the limitations of exercise countermeasures alone, because some individuals have certain congenital or pre-disposing conditions that either make the countermeasures ineffective or unacceptably elevate the risks of flight. Because of the considerable investment of time and resources in the training of air crew and astronauts, imaging studies may be conducted to identify individuals with certain pathology. Plain film x-ray, for example, may be used to identify spine conditions such as spondylosis, spondylolisthesis, and spina bifida occulta in prospective aviators . FurtherThe results of studies on cervical resistive exercise suggest that a self-directed portable exercise device is easy for mobile aircrew to employ and can increase neck muscle endurance and cervical spine range of motion and reduce neck pain frequency in pilots. These results and future studies should inform the resources made available and timing of intervention for pilots to ensure crew health and safety, prevent medical disqualification, and enable mission-readiness. This type of intervention could also potentially prove to be applicable for astronauts and other personnel exposed to similar risks of flight. Further effort is needed to extend enrollment to demonstrate reproducible results in a more varied cohort of patients in both training and operational phases.The wide variety of approaches available to counteract the effects of flight on crew together with the preliminary success of portable, self-directed exercise intervention provide promising relief to personnel putting themselves at risk to carry out their missions in an aerospace environment. Further investigations are required to fully elucidate the scope, risk factors, mechanisms, and anatomical structures involved in musculoskeletal pathology in aviation and spaceflight environments to continue to refine the available countermeasures.All relevant data is contained within the article: the original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.Written informed consent was obtained from the individuals for the publication of any potentially identifiable images or data included in this article.DO’C conducted the literature review. DO’C, SD, and VR prepared the manuscript. JJ, BS, and BSS developed the concept of the project and oversaw its execution and edited and augmented the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plants grow on soils that not only provide support for root anchorage but also act as a reservoir of water and nutrients important for plant growth and development. However, environmental factors, such as high salinity, hinder the uptake of nutrients and water from the soil and reduce the quality and productivity of plants. Under high salinity, plants attempt to maintain cellular homeostasis through the production of numerous stress-associated endogenous metabolites that can help mitigate the stress. Both primary and secondary metabolites can significantly contribute to survival and the maintenance of growth and development of plants on saline soils. Existing studies have suggested that seed/plant-priming with exogenous metabolites is a promising approach to increase crop tolerance to salt stress without manipulation of the genome. Recent advancements have also been made in genetic engineering of various metabolic genes involved in regulation of plant responses and protection of the cells during salinity, which have therefore resulted in many more basic and applied studies in both model and crop plants. In this review, we discuss the recent findings of metabolic reprogramming, exogenous treatments with metabolites and genetic engineering of metabolic genes for the improvement of plant salt tolerance. Environmental stresses (ESs), both abiotic and biotic, negatively impact plant growth and development and reduce crop yields by perturbing metabolic homeostasis ,2,3. SoiHigh soil salinity can cause salt stress (SS), which impairs many critical cellular functions in plants by disturbing various physiological, biochemical and metabolic processes . HoweverSeveral approaches, namely chemical priming and genetic engineering, have been employed to enhance plant SS tolerance ,16. PlanVarious ESs, such as salinity, drought and high temperatures, can lead to the hyper-accumulation of a wide range of metabolites in plants . The tolPMs are essential for the normal functioning of plant cells and are directly involved in various biochemical and physiological processes, e.g., photosynthesis and respiration, providing the energy and the precursors required for the biosynthesis of new macromolecules necessary for developmental processes in plants ,33. PMs Arabidopsis thaliana, trehalose regulates the ionic balance and cellular redox state under high salt concentrations  were up-regulated, and activities of antioxidant enzymes were increased in the transgenic Arabidopsis plants  were up-regulated in transgenic rice plants  were uolerance ,168. TheOver the last decade, different strategies have been used for enhancing ES tolerance, including SS based on metabolomic studies, use of priming agents and genetic manipulation. The applications of metabolomics in various studies have improved our knowledge of the metabolite constituents in many plant species, including the metabolic changes that occur in plants under ESs. The plant response/acclimation to SS through the changes of PMs and SMs enable plants to maintain their minimal growth under such limited conditions. Many studies e.g.,  have sho


Upon the arrival of Rouhani’s government in 2013 and in line with his presidential election manifest, the Ministry of Health and Medical Education (MoHME) initiated a series of reforms, so-called Iran’s Health Transformation Plan (HTP), with three main goals: (a) promotion of financial protection; (b) increasing equity in access to health services; and (c) improving quality of health services. Accordingly, to meet the above mentioned, such interventions as reducing the out-of-pocket payments for inpatient services (from around previous 60%); improving the quality of medical and non-medical services; promoting the natural delivery; increasing the accessibility to specialists in deprived areas , and updating of the relative value units (RVU) of tariffs were introduced. These measures initially increased highly the patients’ and fairly physicians’ satisfaction. However, the plan caused some discontent, particularly among the nurses (Nurses strongly believed that after the HTP, they have been experiencing discrimination in payments in favor of physicians. In addition, extremely low costs services in public hospitals, following the HTP, have led to a heavy workload, intensifying their fatigue and dissatisfaction. There were besides complaints about an increasing violence against nurses, and declined prestige after HTP implementation .In the third step of the HTP, RVUs were revised, in an effort to eliminate the informal payments and realize the values and tariffs of the service. The latter is argued to be the most important reason for the nurse’s dissatisfaction and alleged discrimination.According to the street-level bureaucrats’ theory, nurses act as the ultimate implementers of healthcare policies and are those who translate them into practice. Nurses, as the street-level bureaucrats, communicate with citizens directly during providing, and in this process, they might influence the quantity and quality of health care services by their discretion, which lies in the nature of nursing jobs. Nurses could make decisions at the workplace that in a way might shape the organizations’ policies because citizens experience the final impact of any policy through the benefits enjoyed or losses faced by these bureaucrats. TherefoDuring policy implementation, factors such as the socio-political context, the working environment, personal beliefs and value systems can affect the behavior of nurses. If igno


The presence of the pathological movement of pubic symphysis under normal activities characterises a syndrome know as anterior pelvic ring instability . The truThe induced painful stimuli are usually localised in the suprapubic region and inner thigh areas, but they can also affect the sacroiliac joints and the buttocks. Such activities as walking on abnormal surfaces, going up and down stairs, side lying, sexual intercourse, running and standing on one limb can aggravate the symptoms.Diagnosing the syndrome requires a thorough history and a detailed clinical examination. Provocative tests of stressing the pubic symphysis and sacroiliac joints would elicit irritability, as well as deep palpation to the suprapubic area and symphysis. Clinical examination should exclude such pathologies as inguinal/abdominal hernia, trochanteric bursitis, hip impingement and urologic or gynaecological conditions. Radiographic investigations would help, not only to confirm the suspected diagnosis, but also to exclude other underlying bone pathologies. Acquisition of supine pelvic views may demonstrate bone pathology such as the non-union of previous sustained pubic rami fractures, chronic osteomyelitis and the presence of metastatic disease. However, being static, instability will be missed. Consequently, dynamic stress views are essential to detect instability. The so-called ‘flamingo views’ (single limb standing views) will demonstrate the degree of movement  . The verComputed tomography can delineate bone pathologies related to dysmorphism, osteomyelitis and degenerative conditions of the sacroiliac (SI) joints, whilst MRI can help in the diagnosis of inflammation, oedema, subtle infection, degeneration, bone metastasis and overuse tendon injuries. Interestingly, the dynamic evaluation of the pelvic ring ligaments with MRI has not been investigated as yet.The management of anterior pelvic ring instability should take into account the underlying pathology, as well as any patient’s specific demands and expectations. Non-operative measures include bed rest, physiotherapy (focusing of pelvic floor strengthening exercises), alternation of activities, pain relief strategies , pelvic binders, and guided injections of local anaesthetics with corticosteroids. Of note, these have also been used preoperatively to justify surgery. Interestingly, the methods of guidance vary (fluoroscopy and/or CT), and whilst allowing targeted delivery of substances to the pubic symphysis and sacroiliac joints, there is no robust evidence that one substance is superior to the other. Failure of the above measures leads to operative intervention, in the form of pelvic fusion. Pubic symphysis fusion is usually an open procedure involving debridement of the joint cartilage, bone grafting and plating fixation of the symphysis . The useDespite extensive clinical interest and the availability of a number of related publications, there is still a lack of consensus regarding the incidence, clinical manifestations, pathophysiology, diagnostics, treatment algorithms and final outcome of surgical reconstruction.Journal of Clinical Medicine, focusing on this important clinical entity, with the ultimate aim to further advance our understanding of the pathophysiology of this condition and the results of surgical treatment.Consequently, both clinicians and scientists are invited to contribute their work in the special issue in the


In December 2019, COVID-19 emerged and rapidly spread worldwide. Transmission of SARS-CoV-2, the virus that causes COVID-19, is high; as a result, countries worldwide have imposed rigorous public health measures, such as quarantine. This has involved the suspension of medical school classes globally. Medical school attachments are vital to aid the progression of students’ confidence and competencies as future physicians. Since the outbreak of COVID-19, medical schools have sought ways to replace medical placements with virtual clinical teaching.The objective of this study was to review the advantages and disadvantages of virtual medical teaching for medical students during the COVID-19 pandemic based on the current emerging literature.A brief qualitative review based on the application and effectiveness of virtual teaching during the COVID-19 pandemic was conducted by referencing keywords, including medical student virtual teaching COVID-19, virtual undergraduate medical education, and virtual medical education COVID-19, in the electronic databases of PubMed and Google Scholar. A total of 201 articles were found, of which 34 were included in the study. Manual searches of the reference lists of the included articles yielded 5 additional articles. The findings were tabulated and assessed under the following headings: summary of virtual teaching offered, strengths of virtual teaching, and weaknesses of virtual teaching.The strengths of virtual teaching included the variety of web-based resources available. New interactive forms of virtual teaching are being developed to enable students to interact with patients from their homes. Open-access teaching with medical experts has enabled students to remain abreast of the latest medical advancements and to reclaim knowledge lost by the suspension of university classes and clinical attachments. Peer mentoring has been proven to be a valuable tool for medical students with aims of increasing knowledge and providing psychological support. Weaknesses of virtual teaching included technical challenges, confidentiality issues, reduced student engagement, and loss of assessments. The mental well-being of students was found to be negatively affected during the pandemic. Inequalities of virtual teaching services worldwide were also noted to cause differences in medical education.In the unprecedented times of the COVID-19 pandemic, medical schools have a duty to provide ongoing education to medical students. The continuation of teaching is crucial to enable the graduation of future physicians into society. The evidence suggests that virtual teaching is effective, and institutions are working to further develop these resources to improve student engagement and interactivity. Moving forward, medical faculties must adopt a more holistic approach to student education and consider the mental impact of COVID-19 on students as well as improve the security and technology of virtual platforms. COVID-19 was declared to be a global health emergency by the World Health Organization on January 30, 2020 [Undoubtedly, one of the countries most affected by COVID-19 is the United Kingdom . As of MThe impact of COVID-19 on medical education has been substantial. Medical school attachments often require considerable clinical exposure; however, due to the risk of contracting COVID-19, many medical schools in the United Kingdom have discontinued placements . ConsequThe objectives of this study are to review the advantages and disadvantages of virtual medical teaching during the COVID-19 pandemic using the emerging current literature.medical student virtual teaching COVID-19, virtual undergraduate medical education COVID-19, and virtual medical education COVID-19. Qualitative results from the review were obtained by comparing and summarizing existing evidence and theories from recent literature.A systematic review of peer-reviewed literature on the subject of virtual medical education during the COVID-19 pandemic was conducted from May 2020 to June 2020, consistent with PRISMA  guidelines . ElectroThe quantitative and qualitative studies were chosen based on specific inclusion criteria. The first and foremost criterion was that the study must be published in a peer-reviewed scientific journal. Second, the study was required to present original data assessing virtual medical teaching for medical students, with objectives related to analyzing the effectiveness or perception of this mode of learning. Finally, the included articles reported on studies conducted worldwide between February and June 2020, a period of time central to the COVID-19 pandemic. Due to the shortage of available literature, this review considered any eligible study design, including case reports, case studies, cohort studies, randomized control trials, letters to the editor, commentaries, editorials, and perspectives. The first exclusion criterion was that the article was unrelated to undergraduate medical education. Excluded articles included those focusing on postgraduate medicine and on the teaching of other undergraduate health care professional students, such as dental, veterinary, or nursing students. Moreover, articles that assessed virtual teaching before the COVID-19 pandemic or articles relating to former pandemics were excluded.The search algorithm yielded 92 articles from the PubMed database and 109 articles from the Google Scholar database. After successful removal of duplicate articles, 185 articles were processed to analyze their titles and abstracts, and a total of 68 articles were found to be eligible for full-text screening. Following the full-text screening, a total of 34 articles were included for data extraction. An additional 5 articles were added after manually searching the reference lists of the included articles. Prominent findings from the review are presented in a table under the following headlines: summary of virtual teaching, strengths of virtual teaching, and weaknesses of virtual teaching.In the initial search, 201 articles were found in electronic databases. Following the removal of duplicates, 185 articles were scanned on the premise of title and abstract, and a total of 68 articles were determined to be eligible for full-text screening, of which 34 articles satisfied the inclusion criteria. Manual reviews of reference lists enabled the addition of 5 articles to the review. The findings from the 39 papers reviewed are tabulated in This exploratory review questions the effectiveness of virtual teaching for medical students during the COVID-19 crisis by comparing the advantages and disadvantages listed in all available literature reports, as documented in The COVID-19 pandemic has provided medical education institutions with a unique opportunity to adapt and advance their medical teaching methods. Previously, medical institutions relied upon classroom teaching, such as lectures, for preclinical year medical students, followed by various specialty medical attachments, completed in hospitals, for clinical year medical students . As a reThe analysis of the available literature reveals several advantages of virtual teaching for medical students. First, the development of interactive virtual clinical teaching has been shown to be one of the most effective forms of virtual teaching, ranked by advancement of knowledge, student engagement, and student feedback. Hofmann et al  exploredThe main format of virtual teaching for medical students is through virtual web-based platforms. Virtual web-based platforms consist of webinars, case discussions, reading assignments, and prerecorded virtual scenarios . An advaPeer mentoring during the COVID-19 crisis has proved to be a valuable form of teaching. Peer mentoring involves student-to-student teaching; it helps develop active discussion, exchange of ideas, critical thinking, and collaboration between colleagues . In unceStudent perception of virtual teaching is imperative to understand to deliver effective teaching throughout the pandemic. A study conducted by Sud et al  reportedA significant number of published studies indicated that a major disadvantage of virtual teaching is technical difficulties ,38,42,44Papers by Murdock et al  and SleiThe loss of face-to-face teaching was another significant weakness of virtual teaching. The loss of clinical attachments was referenced by numerous publications ,12,22,34The transition to virtual teaching via the use of web-based medical education platforms presents its own individual disadvantages. Studies by Machado et al  and AtreConcerns regarding preclinical year medical student education were also identified in this review. Studies by Hilburg et al  and MianThe loss of formal assessments is an additional weakness of virtual teaching. A study by Longhurst et al  reportedWeaknesses of virtual teaching identified by medical students included reduced interaction between peers and tutors, reduced understanding of individualized learning needs by tutors, and the difficulties of balancing practical and theoretical skills . VirtualThe literature included in this review includes papers written worldwide. Upon analysis, it is evident that virtual teaching for medical students differs by country and that students may have exceedingly different learning experiences ,30,35,37In developed countries, such as the United Kingdom, Italy, the United States, and Australia, virtual teaching for medical students is a praised method of teaching ,35-37. SIn stark contrast, developing countries such as Nepal, Iran, and Brazil negatively reported on the effectiveness of virtual teaching due to poorly funded and inadequate infrastructures for virtual learning ,24,29,30Virtual teaching for medical students is novel, and the distinct lack of social interaction may increase feelings of isolation, anxiety, and boredom . The lacA study by Hodgson et al  discusseLimitations to be considered in this review include its preliminary and exploratory direction; the literature available for review was restricted due to the emerging nature of COVID-19. In turn, this limitation governed a wider set of inclusion criteria and allowed the acceptance of all types of manuscripts within the review, which may reduce the acceptability of the results to a broader population. Moreover, PubMed was the only legitimate scientific database used, with Google Scholar providing supplemental searches. This limitation raises concerns that some papers relevant to the topic may have been missed. In addition, in the literature analyzed, many studies had small sample sizes; this may decrease the reliability of the findings. A further limitation of this review was the lack of studies that incorporated student perception of traditional teaching in comparison to virtual teaching.Virtual teaching for medical students has enabled medical education to continue despite the effects of the pandemic. The COVID-19 outbreak has provided medical education faculties with the perfect opportunity to develop and further the application and effectiveness of virtual learning for medical students. Medical education faculties should embrace the transition to virtual teaching and continue to develop web-based materials, such as secure web-based assessments and resources with increased student interactivity, to ensure that the most effective and suitable teaching is delivered. Virtual teaching requires significant investment from institutions, and many education faculties worldwide are struggling; institutions should actively seek to share web-based learning materials to improve content and accelerate student learning. Technical challenges and security concerns are inevitable barriers to virtual learning; students and staff members alike should strive to minimize these barriers.


In chronic liver diseases and hepatocellular carcinoma, the cells and extracellular matrix of the liver undergo significant alteration in response to chronic injury. Recent literature has highlighted the critical, but less studied, role of the liver vasculature in the progression of chronic liver diseases. Recent advancements in liver-on-a-chip systems has allowed in depth investigation of the role that the hepatic vasculature plays both in response to, and progression of, chronic liver disease. In this review, we first introduce the structure, gradients, mechanical properties, and cellular composition of the liver and describe how these factors influence the vasculature. We summarize state-of-the-art vascularized liver-on-a-chip platforms for investigating biological models of chronic liver disease and their influence on the liver sinusoidal endothelial cells of the hepatic vasculature. We conclude with a discussion of how future developments in the field may affect the study of chronic liver diseases, and drug development and testing. The liver is the largest solid internal organ in the human body and plays an important role in sustaining regular physiological activities. It has a significant role in metabolism of nutrients and detoxification of exogenous compounds ,2. BecauEfforts to study the role of the vascular component in response to chronic liver disease have many challenges. Due to the multiple cell types contributing to this complex microenvironment, which is composed of multiple regions, gradients, and hemodynamics, in vivo animal models have been at the forefront of liver disease research and have been utilized for pharmaceutical testing and cellular investigations. The primary limitations of these models, however, are the inability to perform as detailed mechanistic studies due to the lack of tunability and to closely investigate the dynamics of disease progression ,6. For tWith recent advances in microfluidics, the inclusion of vascular components in engineered in vitro systems allows the investigation of hemodynamics and the permeability of drugs and solutes through the vasculature to hepatocytes. This incorporation of hepatic vasculature can also benefit hepatocytes by helping them retain their functionality relative to that found in the in vivo setting. Recent literature has demonstrated that the inclusion of a vascular component of liver sinusoid endothelial cells (LSECs), with or without continuous flow and physiological wall shear stress, helps to sustain the correct morphology of hepatocytes in vitro . IncorpoTraditionally, liver models used for in vitro studies have included 2D monolayers and static 3D methods, such as organoids, and spheroids ,13. DynaIn this review, we first introduce the liver microenvironment, including cellular composition, microenvironmental properties, specific cellular roles, and current available cell sources for experimentation. We highlight the oxygen and nutrient gradients inside the liver and how they impact the hepatic vasculature. We then briefly discuss how the stiffness of the liver changes in response to chronic liver diseases. We introduce vascularized liver-on-a-chips categorized under different chronic liver diseases or presentations including fatty liver disease, viral hepatitis, fibrosis and cirrhosis, and hepatocellular carcinoma (HCC). We further discuss how these pathologies have been implemented in vitro, how they impact the hepatic vasculature, and the role LSECs play in disease progression. Finally, we summarize the current applications and the challenges facing future investigations of hepatic vasculature study.The liver’s blood supply stems from the hepatic artery and portal vein ,20. UndeThe extracellular matrix (ECM) of the liver provides mechanical integrity and is critical for cell proliferation, migration, differentiation, and protein expression. As such, changes in the ECM structure and stiffness, usually in response to chronic liver disease and fibrosis, have been shown to regulate vascular structure, integrity and functionality ,23. The The liver lobule is composed of three main zones  that are concentrically segmented based on their proximity to the central vein and nearest portal triads that provide inflow a,b. ThesThe heterogeneous configuration of cells along the liver sinusoid presents various chemical and nutrient gradients. The effect of this gradually changes in functional expression and metabolic activity of liver-specific cells along the gradients rom the portal triad to the central draining vein. Extracellular matrix (ECM) compositions, solute concentrations, oxygen availability, gene expression and metabolism vary across the liver sinusoid, and all play a role in the regulation and function of the specific cells depending on location along the gradient. The overall representation of these gradients is illustrated in Metabolic activity of hepatocytes as measured through the expression of cytochromes P450 (CYPs) runs opposite to both the oxygen and nutrient gradients across the zones of the lobule. These CYP enzymes have been shown to significantly change the efficacy of drugs and provide unique considerations for drug design. Some drugs are activated by CYPs in some cases increasing the hepatotoxicity of the compound. Conversely, the effects of majority of drugs are detoxified and inactive as a result of increased CYP activity ,34. As sThe gradients across the liver zones have shown to alter the properties of LSECs. LSECs have fenestrae (window-like openings in the structure of the cell membrane) to facilitate lipoprotein traffic between the hepatocytes and the sinusoid. LSEC fenestrae are highly variable: size decreases closer to the central vein, but the number of fenestra increases. This variation has been shown to affect transvascular flux across the sinusoidal endothelium, which directly influences the transport of solutes and drugs .The liver is composed of both parenchymal cells, hepatocytes, and non-parenchymal cells, which primarily include liver sinusoid endothelial cells, Kupffer cells, hepatic stellate cells, and cholangiocytes. The surrounding structure of the vascular sinusoid is different than the capillaries of other organs. The liver sinusoid is composed of LSECs that are integrated with liver tissue resident macrophages, called Kupffer cells. The sinusoid is surrounded by a perivascular gap, known as the space of Dissé which includes hepatic stellate cells (HSCs). Surrounding the space of Dissé is the hepatic plate, which is composed of the epithelial hepatocytes.Hepatocytes comprise the major population of the liver and perform many of the liver’s key functions. The hallmark of many liver diseases is direct or indirect chronic damage to hepatocytes. Depending on the selection of the appropriate hepatocyte cell line, varying phenotypic or genotypic states of different liver diseases could be investigated. In this section, we outline the properties of different hepatocytes sources for recent in vitro studies and how they have been used for different purposes.Primary derived hepatocytes from healthy human livers provide consistent, physiological, albumin, urea, and CYP expression consistent through the culture duration ,38,39,40Pluripotent stem cells (iPSCs) differentiated to hepatocytes also showed stable albumin, urea, and CYP450 activity ,47. HoweImmortalized, cancer-derived hepatocytes are commonly used in microfluidic systems due to their relative availability and ease of culture. Several studies summarized the representability of commercially available HCC cell lines based on their gene expression in comparison with primary human HCC samples ,51,52. BHepG2 or C3A HCC cell lines are often used as a proxy for healthy hepatocytes due to the limited availability of commercially available non-tumor derived hepatocyte cell lines. However, one of the primary concerns with the HCC cell lines is the hepatocyte metabolism. The metabolic activity of hepatocytes in a diseased liver is dysregulated, typically shown by reduced CYP expression in HCC derived cell lines . This caTo maintain the ease of culturing HCC derived hepatocytes and allow study of physiologically relevant hepatocyte metabolic activity, researchers developed a subline of HCC cell lines with enhanced expression of CYP3A4 mRNA and CYP3A4-mediated activity such as C3A subline 28 (C3Asub28) . C3Asub2Other HCC derived cell lines include HuH-7 due to its permissibility for Hepatitis C (HCV) infection, allowing the study of HCC and HCV . It alsoThe persinusoidal space between endothelial cells and hepatocytes (Space of Dissé) is composed of liver-specific fibroblasts called hepatic stellate cells (HSCs). Due to activation and collagen/fibrin deposition of stellate cells, the space of Dissé is the best location in the liver to investigate ECM-related diseases such as fibrosis. The process of fibrosis is primarily regulated by HSCs via their differentiation to myofibroblasts. Deposition of collagen/fibrin fibers can increase the stiffness of the liver ECM three to five fold ,56.HSCs in the healthy liver state are responsible for storing liposomes, retinoids, and Vitamin A. Recently, LX-1 and LX-2 cells were developed and commercialized; these cells have been used to investigate hepatic fibrosis, cirrhosis, and fatty liver diseases . Other i3), has also been utilized as a Kupffer cell analog for in vitro study.Kupffer cells (KC) are the tissue resident liver macrophages responsible for the release of many effector molecules in response to liver disease or exposure to exogenous substances. They serve as the first line of defense for pathogens entering the liver and preventing initial viral replication . HoweverLSECs regulate the exposure of hepatocytes to sinusoidal blood. This cell type usually demonstrates fenestrations and endocytosis abilities. LSECs not only function as the permeable barrier between the circulating blood, and resident liver cells, they have been shown to help maintain the native morphology of hepatocytes in vitro and help upregulate functional protein secretion such as albumin, urea, and numerous drug metabolism enzymes ,11. CondImmortalized cells provide several advantages over primary cells: they feature an extended lifespan, are functionally stable, and are a cost-effective alternative to primary cells. Immortalized liver sinusoid endothelial cells, TMNK-1, have been co-cultured with hepatocytes to investigate regulation of growth factors and inflammatory expressions , cytokinIn addition to hepatocytes, stellate, Kupffer, and endothelial cells, bile duct cells and other immune cells such as T-cells, play roles in different disease states in liver. Bile duct cells (cholangiocytes) are the epithelial cells surrounding the bile duct and are responsible for the modification of hepatocyte-derived bile processes regulated by hormones, peptides, nucleotides, neurotransmitters, and other molecules along with intracellular signaling pathways and cascades . CurrentMechanical properties of the extracellular matrix regulate cellular functions, such as functional protein expression, proliferation, mobility, and chemoresistance . The humWhile bulk stiffness measurements of the liver through compression or rheological testing has provided insight to the overall mechanical properties of the liver, recent studies utilizing atomic force microscopy (AFM) have provided more precise measurements of how the stiffness of individual fibers varies spatially across the liver lobules, zones, and fibrotic regions. However, it must be noted that AFM measurements will provide fiber stiffness, which may be different than the compression modulus measurements. Through AFM, it has been shown that stiffness in fibrotic regions are significantly higher (1–6 kPa) compared to healthy regions of the liver (150 Pa) . AlternaCommonly used in vitro models to study chronic liver disease utilize 2D hepatocyte monolayers , isolateHepatic inflammation is the leading factor of tissue damage in the liver and is commonly associated with chronic liver diseases such as viral hepatitis, the progression of non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH), and HCC. Inflammation in hepatocytes is commonly induced with exogenous lipopolysaccharide (LPS) administration either at low (1–2 ng/ml) or high (2–10 ng/ml) levels to produce mild to severe inflammation in in vitro liver models ,94,95. LInflammation has been found to be a significant influence in LSEC function and has been investigated in several vascularized liver-on-chip platforms. Clark et al. found that inflammation induced by LPS upregulated gene expression, such as IL-7, MIP-1a, VEGF-A, and TECK of endothelial cells, which was cultured on polyethylene glycol diacrylate 3D hydrogel scaffolds, mediated pro-inflammatory responses . UpregulDue to the prevalence of obesity and Western diets, non-alcoholic fatty liver disease (NAFLD) has emerged as a common chronic liver disease. It is expected to continue to increase as the incidence of obesity rises . NAFLD rThe effect of ALD and NASH/NAFLD on the liver endothelium has been investigated in several studies. Deng et al. investigated the effect of ALD on EA.hy926 endothelial cells  by perfuViral Hepatitis, primarily hepatitis B (HBV) and hepatitis C (HCV) have long remained one of the more prevalent types of chronic liver disease. While HCV is more common in the United States, Europe and Japan, HBV accounts for roughly 50% of the progression to HCC worldwide ,119. In The very first HBV viral replication study was carried out by Sodunke et al., where a system composed of only hepatocytes were transfected with an HBV-genome cDNA or infected with the viral genome expressed from a recombinant adenovirus . In a diProgressive liver fibrosis with the development of cirrhosis is a feature in the later stages of most chronic liver diseases. In response to chronic liver injury, the common progression of a healthy liver to fibrosis and eventually cirrhosis is a multifaceted process that results in a complex histological mix of nodules and fibrous bands . ChronicAs hepatic fibrosis is primarily regulated by HSCs, many microfluidic and 3D studies focusing on the initiation and progression of fibrosis involve the co-cultures of hepatocytes and HSCs. Leite et al., for example, utilized human hepatic organoids to study the activation and ECM deposition of HSCs in response to cellular damage of hepatocytes . HoweverThe involvement of LSECs in hepatic fibrosis is complex. The fibrotic process is preceded by changes in the liver endothelial cells into a pro-inflammatory, capillarized phenotype. This change leads to further dysregulation of endothelial cells in response to advancing fibrosis. In conjunction with the underlying disease, because the liver function and structure are both considerably altered, this endothelial dysregulation results in compromised drug transport and efficacy within the liver . StudiesJuin et al. and Natarajan et al. demonstrated that changes in liver endothelial phenotype can be solely driven by stiffening of the ECM without the influence of other cell types or contribution of cytokines ,102. BotHepatocellular carcinoma (HCC) is the most common form of liver cancer and the second leading cause of cancer related deaths. Recent findings show that increase in arterialization, and sinusoid capillarization, hypervascularity, and angiogenesis makes HCC detrimental to liver’s vascular health . LiteratIn addition to commonly studied liver diseases, several other chronic liver diseases have a great impact on global health. In this section, we will describe these diseases and identify areas for future investigation. Cholangiocarcinoma (CCA) is a rare type of cancer that originates from the epithelial cells of the biliary ducts in the liver and significantly alters the transport of bile drainage. It exists on the spectrum of neoplasia with HC and occasionally coexists with HCC. Although CCA is rare, it presents a grim prognosis as the 5-year survival rate is roughly 25% . Given tAnother vascular complication of disease that has not been investigated in detail is hepatic angiogenesis in HCC. The hypervascularity and other vascular abnormalities, such as arterialization and sinusoidal capillarization, of HCC tumors results in angiogenesis and the further growth of tumor mass . CurrentLastly, drug-induced vascular damage and dysfunction causes dysregulation of homeostatic functions in the liver by increasing nitric oxide and reactive oxygen species activity . ExogenoStudies of the hepatic vasculature in the context of flow are still limited. Many microfluidic liver-on-a-chip models either neglect the significant role of the hepatic vasculature, or do not investigate the role of chronic liver diseases at all. Even though the very first microfluidic liver-on-a-chip was introduced thirteen years ago , new liv


The development of a new vaccine usually consists of a linear sequence of several steps and lasts many years . The sudThe herd immunity threshold for SARS-CoV-2 has been calculated to range between 50% and 67%, with the assumption of no population immunity, in the absence of any interventions and considering all individuals are equally susceptible and infectious [The different types of vaccines under development include inactivated or weakened virus (virus vaccines), protein subunit or virus-like particles (protein-based vaccines) and replicating or non-replicating viral vector vaccines. ,7. HowevAs of 23 December 2020, after more than eleven months since the isolation of the strain of the new coronavirus, there are 287 vaccines candidates, of which 224 are at the pre-clinical stage of development, sixty-three are being tested in clinical studies on humans and fifteen reached phase three of development and are being tested for efficacy on a large scale , followeTo emphasize the importance of the availability of the COVID-19 vaccine, the European Commission has diversified vaccine portfolios and concluded six different contracts with pharmaceutical companies. These vaccine candidates are in an advanced phase of clinical trials and once the vaccines have been proven to be safe and effective, they will be quickly spread. Furthermore, the different types of development of vaccine platforms will ensure the possibility of different strategies ,15,16. WSeveral official documents provide indications on potential priority groups to consider when designing and implementing vaccination programmes ,17 and aLater on, assuming greater availability of COVID-19 vaccines, vaccination strategies could adapt and also include younger individuals in order to address asymptomatic transmission, that largely contributes to the spread of SARS-CoV-2 and that is most common in younger age segments, therefore limiting the circulation of the virus and indirectly providing protection to the most vulnerable individuals. However, in this second scenario it must be proven that vaccines prevent not only symptomatic severe disease but also infection and its transmission. Moreover, the safety profile should be highly rigorous because of the low risk of young people in case of infection and their longer life expectancy.COVID-19 vaccination strategies should be carefully planned and should account for limited availability, especially early after marketing authorization, posing a great challenge in terms of both prioritization and organization. Of capital importance, the issue to be addressed is COVID-19 vaccine distribution and allocation, which is directly related to ethical considerations regarding equitable worldwide access once the vaccines will be available .At present, there are no definite estimates on the number of doses that will be potentially available and on the timing of supply. In order to design and implement a feasible and efficient vaccination programme, delivery aspects need to be properly taken into account and could include type of settings different from the usual ones, according to the target population . VaccinaThe great attention on the COVID-19 vaccine should not distract the focus from other vaccinations. For example, an adequate coverage against flu is crucial to limit the spread of this respiratory virus, to reduce the impact on healthcare systems and to facilitate differential diagnosis with COVID-19 in specific segments of the population at higher risk of symptomatic and severe disease.In conclusion, before the introduction of one or more COVID-19 vaccines, it is of high importance that policy makers, professional and scientific societies and civil society organizations are prepared with vaccine delivery infrastructure, assessing different scenarios and the impact of alternative vaccination strategies. Lessons learned from the H1N1 pandemic should also be considered. In synthesis, key components for successful policy and vaccination programmes include timely, flexible and evidence-based decisions, robust surveillance systems for COVID-19 disease and monitoring of adverse events following immunization, communication plans and equitable access to vaccination. The promise of the new COVID-19 vaccine is great, but there are further challenges yet to be overcome.


The repair and regeneration of articular cartilage represent important challenges for orthopedic investigators and surgeons worldwide due to its avascular, aneural structure, cellular arrangement, and dense extracellular structure. Although abundant efforts have been paid to provide tissue-engineered grafts, the use of therapeutically cell-based options for repairing cartilage remains unsolved in the clinic. Merging a clinical perspective with recent progress in nanotechnology can be helpful for developing efficient cartilage replacements. Nanomaterials, < 100 nm structural elements, can control different properties of materials by collecting them at nanometric sizes. The integration of nanomaterials holds promise in developing scaffolds that better simulate the extracellular matrix (ECM) environment of cartilage to enhance the interaction of scaffold with the cells and improve the functionality of the engineered-tissue construct. This technology not only can be used for the healing of focal defects but can also be used for extensive osteoarthritic degenerative alterations in the joint. In this review paper, we will emphasize the recent investigations of articular cartilage repair/regeneration via biomaterials. Also, the application of novel technologies and materials is discussed. Cartilage is an important tissue that exists in animal and human bodies but despite other tissues, it does have no nerves, blood vessels or lymphatics . Most caTissue engineering suggests a potential approach which seeds chondrogenic cells on biocompatible scaffolds in order to produce engineered cartilage for the injured one . In prevNanotechnology is defined as the science of manipulation of matter at the nanometer scale and nanomaterials describe materials that have at least one dimension in the nanometer size range. At the nanoscale level, materials behave very differently compared to larger scales and nanomaterials show unique physical and chemical properties in comparison to their bulk form ,14,15. PBiomaterial tools, genetic manipulations, and cell sources have extensively grown in the last two decades, having a positive effect on the expansion of truly functional engineered-tissue . Due to The complex structure of articular cartilage needs to be understood prior to the development of a mimicking construct. The articular cartilage has the main responsibility of transmitting the loads to the related subchondral bone, then absorbing the impact forces, leading to the enhancement of a smooth, low-friction, and sliding motion of that joint . When a The extracellular matrix (ECM), which consists of water, collagens, proteoglycans, and non-collagenous glycoproteins, forms the articular cartilage as a connective tissue. Also, cell-secreted hyaluronan, as well as serum-derived hyaluronan-related proteins which have a covalent attachment to this polysaccharide, are found in the ECM ,23. CollEighty percent of the total weight of articular cartilage is due to water molecules, supporting the elasticity and lubricity as along with the action of transporting nutrients . The choCurrently applied surgical manipulations in cartilage reconstruction consist of marrow stimulation including microplasty, microfracture, and autologous chondrocyte implantation (ACI) . The latThe application of novel methodologies in nanotechnology such as 3-D fiber deposition and electro-spinning has led to the enhancement of nanoscaffold quality . Also, tAccording to reports, a biomaterial can be defined as any nondrug substance with the ability to use in treating or replacing any tissue/organ . This deDifferent research fields such as material sciences, molecular biology and nanotechnology are combined in order to have optimal articular tissue engineering . Also, aNatural materials, which are generally employed in effective and satisfactory cartilage repair, include polysaccharide-based  and protein-dependent scaffolds (fibrin and collagen) ,75. Celll- and d-galactose, which has been commonly utilized as a matrix in cartilage tissue engineering. Numerous evidences suggest that this compound is appropriate for the encapsulation of chondrocytes in particular. In addition to their entrapping role, generation of chondrocyte-derived glycosaminoglycan (GAG) highlights their beneficial application in cartilage research . I. I169]. The generation of zonally-distinct cartilage layers on nanofibers, the inclusion of sacrificial fibers, and the application of ECM proteins in order to prepare nanofibers have been listed as recent advances in cartilage regeneration  which wiBioprinting technology has achieved attention in numerous tissue engineering approaches for its capability to spatially regulate the location of materials, cells and other biological molecules. Though significant developments have been seen in bioprinted complex 3D tissue analogues, the production of hydrogel bioinks with great printability and bioactive specifications should be promoted in order to have an acceptable clinical use. Ávila et al. assessed the biological efficacy of a bioink composed of nanofibrillated cellulose and alginate (NFC-A) for auricular cartilage tissue engineering which facilitated the biofabrication of the cell-laden, patient-specific auricular construct, high cell density and equal cell spreading. NFC-A bioink supports the neo-synthesis of cartilage-specific ECM components and the redifferentiation of hNCs. In addition, the cell-laden NFC-A constructs showed a great shape and size stability as well as an enhancement in proliferation and viability of cells. This confirmed that NFC-A bioink offering suitable printability in a biologically appropriate aqueous 3D environment, turning it to a proper strategy for auricular cartilage tissue engineering  172]..172].Articular cartilage, as a load-bearing tissue, regulates the biomechanical properties of the body via a variety of mechanical and biological properties . HoweverThe most prominent challenge in the use of stem cells for differentiation into the chondrocyte phenotype is the avoidance hypertrophy, which demands biological, chemical and physical regulations . The traOver the last decades, the researchers have developed newly formed articular cartilage replacements. They have attained a worthy comprehension of the physical, chemical, and morphological properties of native articular cartilage. Moreover, they made forward investigations, specifically in the application of cutting-edge printing techniques for imitating articular cartilage tissue. Nonetheless, the investigations should be extended to an accessible, simple and cost-effective approach to the treatment of articular cartilage damage, otherwise, surgeons may follow the traditional approaches for the treatment of patients with chondral defects. Despite developments in the design of scaffolds and bioreactor situations, engineered cartilage constructs remain mechanically inferior to native cartilaginous tissues. Furthermore, the engineered tissues displayed far weaker integration and quality deterioration over time when assessed in laboratory animals.Cartilage tissue engineering needs cells, signaling molecules, and an appropriate scaffold to prepare a new cartilage tissue under special settings. The quick development in material science and engineering has caused progress in the alternative medical approaches for different diseases such as cartilage defects. Nevertheless, cartilage tissue engineering is still in the developing stage. A lot of potential variables in this approach existing and the significant problems remain to be solved. Numerous fields including material science, biology and nuclear transfer are involved in cartilage tissue engineering and expert staffs in methods of cell treatment and polymer synthesis are crucial for the effective application of mentioned methods to produce novel cartilage. The forthcoming investigation in this field should be designed to study and assess the application of tissue engineering technologies as well as surgical methods for cartilage repair in diseased animal models to achieve an improved comprehension of clinically viable design. The progress of a model system for the the investigation of cartilage tissue pathologies is vital.The development of innovative nanomaterials with preferred characterizations could enhance the chondrogenesis of appropriate tissue grafts for the regeneration and repair of cartilage. Recently, some studies performed cartilage tissue engineering, such as nanofibrous scaffolds produced through thermally induced phase separation or electrospinning method, nanosurfaces produced via lithography or acid/base treatments, and nanocomposites produced by integrating of nanomaterials in scaffolds. Nanomaterials lead to produce scaffolds that more closely imitate the natural cartilage tissue in ECM structure. The mentioned process enhances the interaction between the cells, scaffold, and tissue growth. These nanoscale features have displayed achievement in improving cell growth, morphology, differentiation, adhesion, and the production of cartilaginous ECM. An upcoming study in this area should ensure the production of nanocomposites with both biomechanical and biochemical characteristics that imitate the functional and morphological properties of the human cartilage tissue. The produced engineered cartilaginous tissues would be appropriate for final clinical application.


Polymeric nanocarriers (PNs) have demonstrated to be a promising alternative to treat intracellular infections. They have outstanding performance in delivering antimicrobials intracellularly to reach an adequate dose level and improve their therapeutic efficacy. PNs offer opportunities for preventing unwanted drug interactions and degradation before reaching the target cell of tissue and thus decreasing the development of resistance in microorganisms. The use of PNs has the potential to reduce the dose and adverse side effects, providing better efficiency and effectiveness of therapeutic regimens, especially in drugs having high toxicity, low solubility in the physiological environment and low bioavailability. This review provides an overview of nanoparticles made of different polymeric precursors and the main methodologies to nanofabricate platforms of tuned physicochemical and morphological properties and surface chemistry for controlled release of antimicrobials in the target. It highlights the versatility of these nanosystems and their challenges and opportunities to deliver antimicrobial drugs to treat intracellular infections and mentions nanotoxicology aspects and future outlooks. In the frontier of different areas, nanochemistry uses a variety of methods to assemble materials at a nanometer-scale size, with new unique features in respect to the bulk material counterparts, including electronic, magnetic, optical, chemical, and mechanical properties. The interdisciplinarity of nanochemistry involves scientific and technical knowledge from diverse fields such as natural- and material-sciences, engineering, medicine, and pharmacy, among others. Although it is not an easy task, it is aimed at searching for new or existing (bio)materials, understanding their interactions and providing new functionalities towards new products and unexpected applications; these perspectives are encouraging and highly promising, up to the point of being able to generate a revolutionary knowledge of the world at the nanoscale. Within the health care field, nanochemistry offers outstanding opportunities for the design of diagnostic and therapeutic tools; for example, nanoconjugates and nanoplatforms assembled for the controlled and site-specific delivery of active principles with enhanced pharmacological properties against intracellular microorganisms. Intracellular infectious diseases represent a major challenge in health care due to the low specificity of available treatments and the appearance of co-infections and drug-resistant pathogens, which limits the existing therapies ,3,4,5,6.In the last years, the encapsulation of drugs into synthetic and natural polymeric nanocarriers (PNs) has been one of the most explored systems for drug delivery. The resultant nanosystems enjoy high biocompatibility and biodegradability, being very reproducible and amenable for mass production. PNs are usually designed to be stable, have high drug loading capacity and the ability to transport one or more active ingredients , or a combination of therapeutic agents in the same formulation. Such features are very appealing for delivering drugs by different routes and multipurpose clinical approaches ,13,14,15Along with modulation of size, form and physicochemical properties of the polymer, to functionalize PNs with specific ligands may actively drive drugs to site-specific for the efficient drug uptaking, thereby readily reaching therapeutic intracellular levels ,29,30,31In recent years, a large number of drug delivery systems have been described in many medical areas, including intracellular infections ,29,30,33This review provides an overview of the use of different organic nanoparticle precursors and the main assembly methods to produce nanoplatforms of tuned physicochemical and morphological properties and surface chemistry for controlled release of antimicrobials in a target cell or tissue, in a physiological environment, depending on the active principle nature and administration route. It points out the remarkable versatility of these nanosystems and details the facing challenges, as well as the opportunities of the nanoplatforms to deliver antimicrobial drugs to efficiently and effectively treat intracellular infections. It finally mentions some nanotoxicology aspects essential to be considered and prospects in the topic that expect to stimulate advances and new opportunities in this promising field. The efficient treatment of intracellular infections by antimicrobials is highly challenging. Challenges are related to the evasion of intracellular infectious agents by host phagocytic killing mechanisms, the establishment of intracellular survival machinery and the worldwide misuse of antibiotics, which are rising multidrug resistance of pathogens ,41. BesiPNs are an alternative approach extensively studied to overcome some limitations of antimicrobial therapies by encapsulating drugs to improve their ability to enter the cells and release the cargo intracellularly ,45,46. AThey may reduce drug dose and adverse side effects and provide better efficiency and efficacy of therapeutic regimens. By modulating properties and functionality of PNs they can specifically address the target tissue or cell depending on the route of administration .PNs are commonly built by self-assembly of biocompatible and biodegradable polymeric materials to minimize non-specific cytotoxicity on healthy tissues with convenient degradation kinetic profiles and complete metabolization of degradation products ,49,50. AIn nanotherapeutics based on passive targeting, nanocarriers must reach the site of action by physiological or physicochemical changes that occur naturally in the body. They include differences in the pH among tissues (tumor microenvironment), defective vasculature enhanced permeability and retention effect (EPR), capture by the mononuclear phagocytic system and differences in the redox properties of the systems. In active targeting, the surface of nanocarriers is modified to generate affinity with bioreceptors or cellular biomarkers, tissues or organs through ligand-receptor interactions ,53. LigaDrugs are commonly released from nanocapsules rapidly or in a sustained fashion. It depends on the formulation. Drug release can be by diffusion through the porous or polymeric chains of the PNs or by osmotic-, erosion- or degradation-processes. However, natural release mechanisms are migrating towards more sophisticated ones based on physical, chemical or biochemical external stimuli. For example, the response of PNs can be triggered by stimulation with radiation, ultrasound, magnetic fields and temperature. The response can be modulated by changes in the pH and ionic strength, the cellular environment, or by enzymes . Among sNanohydrogels encapsulate hydrophobic and hydrophilic compounds, where the cross-linked network functions as a matrix holding the absorbed liquid medium, which modulates the diffusion of the active principles . In contPNs can be obtained from natural or synthetic polymers, having specific physicochemical features derived from the polymer chemical properties and interactions when assembling the nanostructures. Numerous types of biocompatible and biodegradable polymers from natural or synthetic sources have been reviewed elsewhere ,63,64,65PLGA has excellent biodegradability, biocompatibility, mechanical strength, stability under physiological conditions, low toxicity, and amenability for controlled release. Indeed, there are currently pharmaceutical products based on this polymer, which already have a long history of clinical use . PLGA del-lactic acid), PDLA (poly-d-lactic acid) and PDLLA (poly-d-l-lactic acid). Whereas PLLA has been reported to be highly crystalline, PDLA is entirely amorphous, and PDLLA is amorphous. In contrast, PGA is not chiral and highly crystalline. When copolymers of PLGA are prepared from PLLA and PGA, the resultant polymer has a crystalline character, but whether copolymers are made from PGA and PDLA, the amorphous character of the latter predominates producing an amorphous polymer. If PDLLA and PGA are used, the polymer obtained is amorphous . . 101]. EThe first steps in nanoemulsion preparation using high energy methods are macro droplet formation, deformation and interruption of macro droplets towards nanometric-size ones and surfactant adsorption at the interface to stabilize them by steric effect ,103. AftRecent advances in nanofabrication methods include 3D nanofabrication technology, where the objects are built additively by using cross-sectional layer methods. Besides, updated microfluidic systems such as flash nanoprecipitation (FNP) offer precise control at the nanoscale with multiple applications in the development of PN-based therapeutic systems ,108,109.PNs release therapeutic principles by transporting them from some initial position in the polymer matrix to the outer surface and, subsequently, to the environment in which nanoparticles are found. Drug release follows different release patterns, or a mixture of them, explained by mathematical modeling. Mathematical models allow predicting active principle release rates and their performance to optimize the formulation towards a more precise dosage regimen that minimizes adverse effects and costs. Mathematical models are varied and classified as empirical/semi-empirical and mechanistic models. Empirical/semi-empirical models are purely mathematical descriptions and are not based on any chemical, physical or biological phenomenon; neither reveals factors controlling active principle release and its predictive power is generally low. Yet, they are useful to describe different stages of the active principle release process. In contrast, mechanistic mathematical models are based on real phenomena such as diffusion (based on Fick’s law), degradation and erosion, and are useful in understanding active principle release mechanisms.It is important to note that there is a considerable variety of release mathematical models, including the contribution of Fickian and non-Fickian processes, distribution functions, such as Korsmeyer-Peppas, Higuchi, zero-order, Lindner-Lippold, Ritger-Peppas, Peppas-Sahlin, Weibull, and Monte Carlo technique, among others ,113. HowStimulus-sensitive nanoparticles d can chaMycobacterium tuberculosis, Listeria monocytogenes, and Salmonella typhi), some protozoa , and a few fungi  can survive inside mononuclear phagocytes , the more efficient immune cells to eliminate the microorganisms. Likewise, the intracellular environment protects the microorganisms against attack mechanisms of the humoral immune response, such as antibodies [Infectious diseases are a major health public problem, being the second cause of death worldwide ,116,117.tibodies ,119,120.Some intracellular microorganisms have lost the ability to live outside their host, with whom they always have intimate and dependent relationships ,122,123.Antimicrobials are powerful drugs to fight infectious diseases, but there exist relatively few options in the market ,126. IndThe development of drug resistance has occurred on a large scale over time, reducing the effectiveness of treatments. Microorganisms produce resistance by acquiring the expression of resistance genes or by selecting microorganisms that express them, which gene acquisition can take place spontaneously or by horizontal transfer usually in response to drug exposure ,133,134.Drugs used for the treatment of intracellular infection diseases are typically administered by oral, intravenous (IV) and inhalation routes. Depending on the administration route, drugs are exposed to several challenges due to the existence of biological barriers in the human body. The biological barriers exist to maintain homeostasis, which prevents any foreign substance and pathogen ,163,164.IV is an invasive administration route with the inconvenience of having high costs and produce adverse effects. The major barriers to the IV route treatment include the liver and the spleen, which together are the clearing organ, forming a dynamic barrier to keep harmful substances out of the body. The organs in their capillaries have many mononuclear phagocytes, which constitute the reticuloendothelial system (RES) ,162,164.Junctions between the cells in the arteries, veins, and capillaries form another IV barrier that keeps our blood flowing but makes it difficult for the drugs to cross, which is a phenomenon known as extravasation ,162. FolOral is the most convenient method of drug administration, as less sterility of the therapeutic agent is required and it is pain-free. Yet, the effectiveness of drugs administrated orally is reduced by both acidic pH and action of enzymatic digestion in the stomach ,169. OncThe pulmonary route of administration is relatively complex because the respiratory tract develops mechanical, chemical and immunological barriers to keep inhaled therapeutic agents out of the lungs and remove or inactivate them once they go inside. The therapeutic agent needs access to the large lungs’ epithelial layer but must pass numerous airway bifurcations where it could be deposited ,172. To Administration by the skin is increasingly being used in the application of therapeutic agents due to it being a less invasive route. The skin is the first line of body defense, preventing the entrance of chemical and biological agents. The epidermis is composed mainly of a layer of keratinocytes (95%) and a minority of melanocytes (melanin production cells) and dendritic cells (involved in the immune response), whose composition absorbs lipophilic compounds through the skin. The substances traverse the barrier by transcellular, intercellular, and transappedageal processes (diffusion via the hair follicle’s sebaceous and sweat glands) ,175. TheIn contrast, large molecules have a physic restriction to penetrate lipid channels. Diffusion through the hair follicle, sebaceous and sweat glands is an alternative pathway to cross SC because their structure provides a niche for mechanical accumulations and storage of substances. Additionally, the easy access to the bloodstream and lymph makes the hair follicle a potential target to administrate therapeutic agents for local or systemic applications, as follicle size is one of the main characteristics influencing the penetration by the hair ,175.Eukaryotic cells are sub-compartmentalized in membrane-bound structures known as organelles , being the target of the intracellular pathogens that survive off proteins and lipids from organelles within the host. Nanosystems to treat intracellular pathogens have the challenge of delivering drugs at the intracellular level in the cytosol or through the specific organelles. The pathogenesis process takes place by invasion, survival, replication, and exiting the host cell. The invasion involves the steps for extracellular to intracellular space transition. After the invasion, some survive by altering antimicrobial mechanisms and others create a compartment, which is impervious to the microbicidal mechanisms of the host cell. For all intracellular pathogens, surviving in a permissive cell leads to replication with an increase in intracellular microbial loading , and theMycobateria tuberculosis [Salmonella [Coxiella burnetii [Histoplasma capsulatum [Many intracellular pathogens evade the host immune response causing disease by residing and replicating inside the host cells, primarily macrophages. One of the main strategies of microorganisms such as bacteria, protozoa and fungi is the manipulation of the organelle “phagosome” involved in phagocytosis, the main mechanism of defense of macrophages ,124,177.rculosis , Salmonelmonella , Coxiellburnetii  and Histpsulatum  are typiLegionella pneumophila [Brucella [Toxoplasma gondii [Chlamydia locates into the Golgi apparatus [The second group uses proteins and lipids from host cells to replicate and some of them can convert the phagosome-lysosome into an organelle similar to Endoplasmic Reticulum (ER) and Golgi apparatus. Whereas umophila , BrucellBrucella  and Toxoa gondii  can locapparatus ,182,183.pparatus ,185. EacNanosystems’ surface modification is a good alternative to deliver drugs in a site-specific way. After internalization by endocytic pathways, the main challenges are to reach out to the cytosol by escaping the endolysosome compartments avoiding premature drug release and the acidic pH inside the organelle. One way to achieve disruption of the endolysosome structure and thus, drug release in the cytosol is through pH-response polymers ,182. VirListeria monocytogenes cause mitochondrial morphological changes such as mitochondrial fission by secreting a pore-forming toxin to overcome ROS [The role in the regulation of different functions influencing the intracellular survival of pathogens evading host immunity makes the mitochondria another important targeting organelle. For instance, mitochondria produce ROS that can affect intracellular pathogen survival ,185. Liscome ROS ,186 is acome ROS ,187,188.come ROS .Targeted intracellular drug delivery is a major challenge to succeed in nanotherapeutics, where the type of endocytosis process is particularly important to consider for killing the pathogens. In this context, phagocytosis by macrophages plays such an important role in the connection of innate and adaptive immunity by expressing pattern recognition receptors (PRR) on their surface to identify parasites and pathogen-associated molecules, favoring the protection of cells against attack of foreign agents. Macrophages expressing plasmatic membrane receptors mediate important interactions with components of host and with foreign microorganisms to improve host defense, inflammation, homeostasis and modulation of immunity, which made them relevant targets of nanotherapeutics for intracellular infections.Receptor-mediated endocytosis is another important strategy widely employed by researchers to design improved nanotherapeutics by using ligands attached to the nanoparticles that can be recognized by the cells for a site-specific targeted therapy . In thisOne of the most exciting strategies of functionalized PNs is the use of natural or synthetic polymers with different targeting ligands to provide specificity for organs or tissues, thus improving the uptake of nanoparticles from, for example, the gastrointestinal tract when oral administration is employed, the preferred drug administration pathway among the population. This strategy is relevant, as ligands and certain polymers have shown enhancing oral absorption ,199. MorNew efforts to improving antimicrobial therapy include encapsulation of antimicrobial peptides and oligonucleotides in PNs, encapsulation of natural products with antimicrobial properties and nanocrystals of antimicrobials stabilized in a polymeric matrix ,201,202,Functionalization of PNs with ligands using PCL as polymeric- and building hybrid-platforms with metallic nanoparticles and mesoporous silica has been explored in the drug delivery of antimicrobials and viral therapy ,215,216.Curcumin is an active principle with multiple pharmacological activities, but its high hydrophobicity restricts its aqueous solubility and applicability ,219,220.Natural products with antimicrobial properties encapsulated in PLGA PNs is a trend in recent studies. For example, Rosin acids (RA) derived from coniferous trees and composed of a mixture of hydrophobic diterpene carboxylic acids, have been used to treat skin infections. Santovito and collaborators recently nanoencapsulated RA within PLGA-PEG PNs and proved its antimicrobial activity against different Gram (+) and Gram (−) pathogens . NanoencHyaluronic acid (HA) is a polysaccharide obtained from natural sources found in microorganism membranes and animal tissues having multiple properties, including inhibition of tumor growth, wound healing and anti-inflammatory properties. HA has been explored extensively in biomedical applications and drug delivery ,223. It S. aureus, E. coli and B. subtilis showing the strong killing effect of cationic groups from PEI polymer in binding and precipitating some cellular materials such as lipid and nucleic acids [M. tuberculosis in infected macrophages [Antimicrobial activity of (HA)-PEI nanogels against Gram (+) and Gram (−) bacteria strains were evaluated in ic acids . Encapsurophages . In generophages ,229,230.Another tendency in recent studies about site-specific therapies for infected macrophages inducing an immune response is exploiting the C-type lectin superfamily receptors. They are an essential target to be used in antimicrobial therapy because of their expression on the surface of many intracellular pathogens ,210,231.The extensive biological research has contributed to identifying new therapeutic targets. For example, the neonatal Fc receptor (FcRn) is a recently approved patent for improving the oral administration of nanotherapeutics (Patent number: WO2020086871A1) ,235. It Extensive studies in nanomedicine based on the development of new therapies to treat different diseases have been done during the last decade. A growing number of nanosystems have received regulatory approval and the majority were safe, biocompatible and effective. Due to the use of different materials, solvents, shapes, targeting ligands and methods of manufacture, it is imperative to evaluate the toxicity, biodistribution and pharmacokinetics of the nanosystems to reach a safe and effective therapy towards the translational clinic. Even though the PNs have demonstrated significant advantages in the delivery of drugs against intracellular pathogens, they present disadvantages such as the use of organic solvents in their fabrication process, physiological barriers to overstep, poor biocompatibility, immune response activation and cytotoxicity. Understanding the physicochemical characteristics of nanoparticles and how these characteristics affect their interaction with the body is imperative to overcome limitations and in establishing strategies to improve therapy efficiency.The pharmacokinetics of PN-encapsulated drugs can be affected by size, shape and materials composition, which impact both the degradation of nanosystems and drug release kinetics. Xu et al. demonstrated that an increased superficial area/volume ratio decreased the drug rate of biodegradation, being lower in rod-shaped (30 days) and tablet (30 days) nanosystems and faster in film (19 days) and microsphere-shaped ones (16 days). The encapsulated drug in films released five times faster than in microspheres . The shaPhysiological pH can modulate the polymer degradation rate, affecting the drug release rate, i.e., strong acidic and basic environments accelerate PLGA degradation, thus increasing the drug release rate . AdditioOne crucial issue to improve PNs’ biodistribution is the capacity to cross physiologic barriers, which can be modulated by the physiochemical characteristics of the PNs. The main physiological barriers that the PNs need to overstep are the cells belonging to RES and MPS, especially the macrophages of RES, which promote a high biodistribution of PNs in the liver or even cause a rapid elimination of them from the body.The recognition of PNs by these types of cells can be modulated by size, shape, surface charge and hydrophobicity/hydrophilicity of PNs because such characteristics regulate the interaction with physiological proteins and the protein corona formation. The type of proteins from the protein corona determine the PNs biodistribution or their fast elimination from the body. PNs size, shape, and superficial charges affect the number of plasma proteins that adhere to the nanosystems surface and, therefore, the cells uptaking. PNs with a positive charge, big size and nanorods shape adhere more proteins than smaller nanosystems and they are easier uptaken by cells b,c,e 26,268,269.Similarly, the chemical polarity of the nanosystem promotes cellular uptake, which is also involved in the protein and lipid adhesion. For instance, hydrophobic nanosystems adhere a higher amount of plasma proteins and have a high affinity for the hydrophobic lipids of the membrane cell, increasing the probability of cellular uptake g. PEGylaSize plays an essential role in the biodistribution of PNs. PNs of smaller sizes have the tendency to aggregation, decreasing their capacity of biodistribution. PEGylation reduces the PNs’ aggregation, avoiding their accumulation in non-targeted organs. Biodistribution of nanosystems administered by the intranasal route is highly particle size-dependent. In general, inhaled PNs with aerodynamic diameters between 0.5 and 5 µm are deposited in the central and distal tracts, while those with a size >5 µm are trapped in the upper airways  and those with size <0.5 µm are mostly exhaled. Besides, modification of the PNs’ surface may help to modulate the deposition pattern in the lungs . Ungaro PNs can be modified with bioadhesive materials such as PVA, PEG, and vitamin E-TPGS, among others, to improve the adhesion interactions and enhance the internalization into the intestinal cells as well as the ability to escape from efflux pump proteins ,273. A hThe main option to regulate the biodistribution through a target cell or tissue is by targeting ligands. Resident macrophages are present in each organ from the body, having different characteristics and functions. They adapt to the tissue location, which impacts on their biological function. The expression of different receptors characterizes the macrophage population of each organ. For example, lung macrophages in mice  have high expression of mannose and siglec-F receptor and low expression of F4/80 receptor as compared to peritoneal macrophages that present an intermediate expression of F4/80, low expression of mannose and no expression of siglec-F receptor . MannoseThe specificity of targeting ligands can be affected by its density and orientation onto the nanoparticle surface, thus, impacting its biodistribution. Functionalization methods may modulate the density and orientation of molecules or by masking the recognition region from cell bioreceptors . For insBefore advancing towards in vivo applications, it is necessary to test possible toxic effects of PNs on human health related to their physicochemical properties. One of the main purposes is to study the nanosystem immunotoxicity. Reports have associated the high level of proinflammatory cytokines upon nanosystem treatment with low therapeutic efficacy, side effects and toxicity. The level of toxicity of PNs depends on the size and administration dosage. Negative charged PNs can activate the immune response, whose activation is related to pseudoallergy; and increased hemolysis. Neutralizing the charge by surface modification of the nanosystem with other polymers or materials prevents binding of protein corona to the nanosystem and then activation of the immune response. Tirtatmadja et al. observed that a variation of the protein corona composition at the nanosystem affected the production of IL-8 and TNF-α inflammatory cytokines . Benita Hydrophobicity properties and size influences PNs’ toxicity. PNs with a high hydrophobicity induced acute respiratory toxicity upon single-dose administration . SmallerDespite the multiple reports highlighting the properties of PEGylation to reduce an immunogenic response, many publications have demonstrated that PEGylation may cause immunogenic effects in some individuals, inducing the production of antibodies, which enhance blood clearance, reduce the efficacy of nanotherapeutics and increase side effects ,290,291.Attempts to fight intracellular infectious agents have given us important lessons, bringing out how urgently needs to be the efforts to develop alternative antimicrobial therapies that manage to increase antimicrobial efficacy and decrease microbial resistance as compared to conventional antimicrobial treatments. In this context, PNs have emerged as a nanoplatform having broad prospects for the development of highly promising site-directed antimicrobial therapies for the management of intracellular infections whose products on the market expect to be established shortly, as judged by the number of research publications and patents currently granted in the field.Recent advances in nanoparticulate formulations, including hybrid strategies, nanoencapsulation of natural products, targeting ligand-based formulations and smart materials, have shown to allow tuning the bio-physicochemical properties of PNs for enhanced antimicrobial efficacy. However, there are still many challenges to face to improve PN-based antimicrobial technology towards scaling-up at the industrial level and reach the market. For example, PNs captured by the immune system hinders the site-specific drug delivery facilitated by highly selective targeting ligands leading to ineffective internalization. Nanosystems toxicity and nonspecific biodistribution limit in vivo practical applicability. Furthermore, proper standardization is not trivial both in vitro and in vivo, which limit systematic comparative studies. Therefore, it is necessary to continue researching to advance the limited understanding of the fundamental processes of PNs in and out of the human body, given the multiple interactions of antimicrobial nanotherapeutics among them, with the protein corona and with the vast diversity of organs, tissues and cells on their journey from administration to therapeutic targets.A higher number of standardized production methods, validated studies of toxicity, bioequivalence, clinical studies, and the establishment of reference materials may impact on gaining a better knowledge of the antimicrobial nanotherapeutic systems and their associated pro and contra, in the way to creating products that ensure quality, safety and efficiency. The final balance of this process defines the scope of PN-encapsulated drugs, to quickly reach high acceptance in the market, thereby offering added value as compared to conventional antimicrobial therapies.