Background: Sudden changes in clinical practice and the altered ability to care for patients due to the COVID-19 pandemic have been associated with moral distress and mental health concerns in healthcare workers internationally. This study aimed to investigate the severity, prevalence, and predictors of moral distress experienced by Australian healthcare workers during the COVID-19 pandemic. Methods: A nationwide, voluntary, anonymous, single time-point, online survey of self-identified frontline healthcare workers was conducted between 27th August and 23rd October 2020. Participants were recruited through health organisations, professional associations, or colleges, universities, government contacts, and national media. Results: 7846 complete responses were received from nurses (39.4%), doctors (31.1%), allied health staff (16.7%), or other roles (6.7%). Many participants reported moral distress related to resource scarcity (58.3%), wearing PPE (31.7%) limiting their ability to care for patients, exclusion of family going against their values (60.2%), and fear of letting co-workers down if they were infected (55.0%). Many personal and workplace predictors of moral distress were identified, with those working in certain frontline areas, metropolitan locations, and with prior mental health diagnoses at particular risk of distress. Moral distress was associated with increased risk of anxiety, depression, post-traumatic stress disorder, and burnout. Conversely, feeling appreciated by the community protected against these risks in healthcare workers. Conclusions: Safeguarding healthcare workforces during crises is important for both patient safety and workforce longevity. Targeted interventions are required to prevent or minimise moral distress and associated mental health concerns in healthcare workers during COVID-19 and other crises. The COVID-19 pandemic has led to growing international awareness regarding the prevalence of moral distress and psychological symptoms amongst frontline healthcare workers (HCWs) . Moral dWhile the effects of moral distress seem to be similar to those resulting from psychological distress and burnout, studies examining the relationship between these concepts in frontline HCWs are limited ,18,19. TFrontline HCWs were invited to participate in a nationwide, voluntary, confidential, online survey between 27 August and 23 October 2020. The recruitment period coincided with the second wave of the pandemic in Australia, with most cases arising in Melbourne, in the Australian state of Victoria [https://covid-19-frontline.com.au/), which held the online survey link. Online consent was acquired from participants prior to commencing the survey, and each respondent could only participate once. The survey included seven sections  with 95% confidence intervals (CIs) with statistical significance declared at Most participants were female  with an even spread of age ranges . ParticiMost participants somewhat or strongly agreed with statements regarding concerns about patients not receiving care due to scarcity of resources  and excluding family from the bedside of patients infected with COVID-19 went against their values as HCWs , working in metropolitan areas , and those with a pre-existing mental health diagnosis . ParticiIndependent predictors for being concerned that wearing PPE would limit the care provided to COVID-19 patients included living in the state of Victoria (compared to other states) or currently working with COVID-19 patients . IndividParticipants who worked in primary, community care, and aged care (compared to ED), those living in metropolitan areas , who had pre-existing mental health diagnoses, or who desired more training regarding PPE or managing patients with COVID-19 (compared to those who did not) were significantly more likely to be worried about burdening their co-workers if they needed to quarantine . Nurses Independent predictors for believing that excluding family from the bedside of COVID-19 patients went against their values as HCWs included female gender , having a pre-existing mental health diagnosis, and indicating need for more training in care for patients with COVID-19 or use of PPE . Non-medOlder participants (compared to those aged 20–30 years) were significantly more likely to believe that the community was worried that HCWs would spread the virus to others . People Independent predictors for believing that the community was appreciative of HCWs during the pandemic included: age, occupation, receiving PPE training, and being confident using PPE. Participants aged 31–40 years (compared to aged 20–30 years) and nurses (compared with doctors) were significantly less likely to believe the community was appreciative of HCWs during the pandemic. Participants who were confident using PPE, trained in using PPE, and participants from other health roles (compared to doctors) were more likely to believe the community was appreciative of HCWs during the pandemic.Being concerned that wearing PPE affected their ability to care for patients with COVID-19 or being worried about letting down colleagues if they needed to quarantine were significant, independent predictors for adverse mental health outcomes on all scales except the personal achievement domain of the burnout scale . Being wTo our knowledge, this is the largest, multi-professional study globally to investigate moral distress and mental health outcomes in frontline HCWs during the COVID-19 pandemic. We have identified personal and work-related predictors for experiencing certain facets of moral distress, as well as demonstrating that prior mental illness, working in primary care, community or aged care, working in metropolitan areas, and desiring more training were predictors of experiencing moral distress. Experiencing moral distress was associated with broad, adverse mental health outcomes, whereas believing that that the community was appreciative of healthcare workers during the pandemic protected against these outcomes. Consistent with previous studies ,19, our Participants were broadly in agreement with three of the four indicators of moral distress relating to resource scarcity, exclusion of family, and perceived letting down of overstretched co-workers if quarantined. Fewer participants agreed that PPE usage limited ability to care for patients with COVID-19, though this was still a concern for a third of participants. Although most agreed that the community was concerned about HCWs spreading the virus, the vast majority felt that the community was appreciative of HCWs.Overall, those with prior mental health diagnoses, primary, community, and aged care health workers, those living in metropolitan areas, and those who indicated the need for additional training were consistently more likely to provide answers indicative of moral distress. These findings may relate to inadequate organisational preparedness and resource availability for staff working in primary, community, and aged care settings. A report prepared by the Aged Care Quality and Safety Commission echoes these concerns and characterised the overwhelming of aged care facilities with one provider stating: ‘We already had a COVID-plan, but we didn’t really prepare for the avalanche of it all’ . AlthougNurses and allied health workers relative to medical staff and ICU staff and other frontline areas relative to ED workers were frequently identified as having reduced odds of reporting moral distress. These results are somewhat surprising given professional autonomy is a frequent predictor of moral distress ,33 and iIn contrast to some prior studies of baseline moral distress in Iranian , CanadiaEndorsing indicators of moral distress was frequently found to be independently associated with moderate to severe symptoms of mental illness on all the validated mental health outcomes tested in the current study. Moral distress has been linked to increased turnover within organisations and attrition of HCWs ,43; failThe demands placed upon HCWs during a time of crises can instil in some workers a sense of meaning or purpose. Qualitative interviews with HCWs in the aftermath of the Haiyan typhoon in the Philippines identified altruistic motives as a means of finding acceptance and control in their circumstances . In the Our findings reveal that HCWs in certain frontline areas, metropolitan locations, and those with prior mental health diagnoses were disproportionately impacted by moral distress during COVID-19. The causes for these disproportionate impacts are likely multifactorial and relate to resourcing shortages, concentration of caseloads, and known vulnerable demographic factors. Although some of these factors, such as case distribution, are uncontrollable, these results are indicative of where targeted efforts can be made to mitigate moral distress and associated adverse mental health outcomes.Due to the pandemic, many hospitals and healthcare organisations set up wellbeing supports, which were previously either not available or severely limited . Given tThe survey included participants from a wide range of healthcare professions and represents the experiences of HCWs across different frontline specialities. The majority of participants in the current study were women, which is consistent with data from both the Australian Institute of Health and Welfare and the Australian Health Practitioner Regulation Agency demonstrating that 75% of the Australian health workforce is female ,53. Due Due to the spontaneous and unexpected nature of the COVID-19 pandemic, no baseline data regarding moral distress in non-pandemic times had previously been collected from a large cohort of Australian healthcare workers. The design of the survey as a single point-in-time data collection was chosen to minimise burden on HCWs; however, future research is required to provide longitudinal follow-up. In the interests of brevity and to focus on COVID-19, we generated questions relating to specific factors of moral distress. These questions targeted similar dimensions of moral distress to those included in longer, validated scales such as the Measure of Moral Distress for Healthcare Professionals (MMD-HP), such as exclusion of family, acting in accordance with personal values as a HCW, and resource scarcity negatively impacting patient care . AdditioThis large-scale survey provided an insight into predictors of moral distress and its correlation with mental health outcomes in HCWs. Given that a healthy workforce is pivotal to effective healthcare service delivery, recognising and identifying moral distress and its downstream effects as well as promoting the development of targeted interventions and evidence-based policies will contribute to the cultivation of moral resilience in HCWs at workplace and community settings.


Management and successful use of vascular access are critical issues in pediatric patients affected by malignancies. Prolonged course of disease, complex and various treatment protocols require long-lasting vascular access providing adequate tools to administrate those therapies and to collect routine blood sampling without painful and repeated venipuncture. For these reasons, central venous catheters are currently an important component in pediatric onco-hematological care, with a direct influence on outcome. Indeed, there are peculiar issues  which must be well-known in order to improve the outcome and the quality of life of children with cancer. Although cancer is a rare condition in children, it represents the second most common cause of death in patients older than 12 months . FurtherIn 1973 Broviac et al. . Gyves aThe aim of this paper is the provide an updated review of the general recommendations for VADs management. Patient’s requirements are the first and most important issue to consider for the choice of vascular access device (VAD), taking also into account expected duration, type and previous history of onco-hematology treatment. There is a large and established consensus that chemotherapy medication should be delivered through an adequate central venous access, in order to reduce to lowest terms the risk of infusion-related injuries. This statement must be applied also for supportive care issues, as well as for management of advanced cancer stages with palliative measures .One of the main concerns for specialists treating onco-hematological children is the high volume and number of medications (either drugs or blood components) to administrate via the catheter. As a consequence, the erroneous idea of “the bigger lumen, the better performance” and “more lumens, easier management” lead to demand for large bore and/or multiple lumen catheter for all patients. Regarding catheter’s caliber, it has also been demonstrated that bigger catheters present higher risk of venous thrombosis; moreover, an outsized catheter requires venotomy during the placement procedure. For the above-mentioned reasons, the size of the device should be based on the diameter of the target vein measured with ultrasound, especially in cancer patients, who are more prone to coagulation disorders  ,16,17,18Instead, multiple lumen catheters are indicated in patients undergoing intensive treatments or stem cell transplantation, considering the risk of undesirable reactions due to drug interacting during administration of multiple intravenous medications and solutions  . For thoAs a general rule, short-term catheters should be chosen in emergency settings  and only for intrahospital use. Thus, most children require long-term venous access for discontinuous and intra/extrahospital use .Toddlers/younger children, who are less tolerant to repeated and painful punctures, and patients undergoing intensive and prolonged chemotherapy regimens could benefit from a partially implanted/tunneled VAD (PI-TVAD), which is easy to handle and allows for repeated infusion/blood sampling without pain. Among their disadvantages are reported the following: disturbed body images, need for exit-site caring with regular dressing, limitations on physical activities . Adolescents/young adults or children with discontinuous therapies  are eligible for totally implanted devices (TID) such as port catheters. These devices do not necessitate local care and do not limit any physical activities, preserving patients’ body image; however, their access (through skin puncture to the reservoir) may be more painful or difficult (in obese patients) and their removal necessitates of surgical procedure. Therefore, TID are recommended in patients requiring intermittent and prolonged use while PI-TVAD are indicated for prolonged but continuous/frequent vascular access . It is nowadays well-known that peripherally inserted central catheters (PICC) represent a versatile, durable and accessible long-term vascular access in pediatric population, due to improvements in materials (III/IV generation power injectable polyurethane), quick placement procedure  and good patients’ compliance. In children with cancer, PICCs are a good solution for patients who necessitate adequate vascular access and could not undergo to general anesthesia  as well as for patients who do not stand PI-TVAD coming out for the chest ,28,29. TOver recent years, evidences discouraging open venous cut-down (OSC) as primary technique for VADs placement have been reported, especially in children with cancer. This procedure should be considered a historical “legacy” of pediatric surgeons of last century ,34,35. ERecently minimally invasive procedures for catheter placement have become the gold standard in adult patients. Though the evidence in pediatric patients is still debated, most studies suggest that ultrasound guidance should become the main technique of venipuncture also in children and neonates ,33,40. IRecommendations from AIEOP for VADs positions are reported in Considering the prolonged course of the disease and the significative improvement in survival rate for children with cancer, strategies to prevent early and long-term complications and to preserve vascular assets directly influence outcome and quality of life for these patients. It is mandatory to acknowledge all the possible scenarios, to adopt preventive strategies and overcome eventual difficulties through prompt recognition and appropriate management.As previously mentioned, minimally invasive ultrasound-guided procedures for VADs placement totally changed the approach to vascular access, especially in pediatric patients. With sufficient training and knowledge of devices available  and technique, ultrasound consents high percentage of first-time success and allows diagnosis of preexisting vascular anomalies , reducing the incidence of periprocedural complications  . MoreoveCatheter dislodgment and/or tip migration may lead to malfunction of the device (either in infusion or in blood draw) and, in worst cases, to complete removal. Pediatric patients undergoing chemotherapy and/or high dose steroids are more prone to these complications for various reasons, such as reduced healing ability and coagulation, increased infection, skin and subcutaneous tissue disease (like graft-versus-host disease). Different approaches to reduce these events have been described, such as the use of non-cuffed third generation polyurethane , secured with both suture-less devices and subcutaneously anchored securement systems (SASS) ,44,45. URecommendations from AIEOP for VADs management are reported in Catheter removal is widely considered a neglected procedure, frequently inaccurately performed by skilled staff members in different environments , according to personal or institutional experience, and not always following evidence-based protocols. Nevertheless, in the case of patients with long-term cuffed devices , catheter removal represents a “real” surgical procedure with non-negligible risk of complications  ,50. For In recent years, many improvements in terms of materials, techniques and care have been described; however, despite many protocols and guidelines being reported over the last decades, there is far less evidence for pediatric patients than for adults, especially onco-hematological ones. Appropriate knowledge and skilled teams are mandatory in order to address issues regarding indications, placement and removal techniques, choice of the devices, catheter care and possible complications in such a peculiar population.


Manifestations of an atypical variant of medulloblastoma of the posterior fossa in extra-axial locations have been reported, and key questions concerning its interpretation have been raised previously. This review illustrated the clinico-radiological and histopathological features of the posterior fossa extra-axial medulloblastoma and described possible management strategies. We thoroughly reviewed all atypical anatomical locations of medulloblastoma reported within the posterior fossa and extra-axial spaces. The main characteristics of diagnostic imaging and histopathological results, primarily the distinctive radiopathological characteristics, were summarized to distinguish between intra- and extra-axial medulloblastoma, or pathologies mimicking this tumor. Most cases of posterior fossa extra-axial medulloblastoma have been reported in the cerebellopontine angle, followed by the tentorial and lateral cerebellar locations. The dural tail sign, which is commonly observed in meningioma, is rarely seen in intra- or extra-axial medulloblastoma and might be associated with other benign or malignant lesions. In addition to magnetic resonance imaging, the proposed new imaging techniques, including advances in modern neuroimaging modalities, were discussed, as potentially efficient modalities for characterizing extra-axial medulloblastoma. Radionuclide imaging and magnetic resonance perfusion imaging are practical alternatives to limit the number of differential diagnoses. We believe that medulloblastoma cases are likely under-reported because of publication bias and frequent tumors in unusual locations. Addressing these issues would help establish a more accurate understanding of this entity. Medulloblastoma is a common brain tumor; therefore, understanding its variations is crucial.Neuroimaging is helpful in the preoperative neuroaxis evaluation and postoperative assessment of medulloblastoma.Identifying and categorizing metastatic disease during diagnosis is paramount for effective therapy.Deciphering this challenging diagnosis can reflect positively on a patient’s prognosis.Medulloblastomas, which are undifferentiated embryonal neuroepithelial tumors, originate from primitive multipotent cells of the cerebellum and spread to the germ cell migration tract . Other sNotably, medulloblastomas are common malignancies in pediatric patients, accounting for 25% of all brain tumors of childhood, typically arising from the cerebellar vermis. In contrast, adulthood medulloblastomas are typically observed within the paramedian region or laterally within the cerebellar hemisphere, and account for < 1% of primary brain tumors among adults [Recently, advanced neuroimaging techniques have revealed the presence of medulloblastoma in extra-axial locations , 2. The Radiologically, medulloblastoma appears as a contrast-enhanced hyperdense region on a computed tomography (CT) scan, often compressing the fourth ventricle; iso- to hypointense on T1-weighted magnetic resonance (MR) images; and hyperintense on T2-weighted MR images . MR specCombined therapy, consisting of a maximally radical surgery followed by chemotherapy and adjuvant irradiation of the entire central nervous system, is typically recommended . PrognosWe searched for articles published in PubMed until August 2020. Search strings consisted of a combination of the following terms: “medulloblastoma,” “extra-axial,” and “exophytic growth.” The literature search was conducted by three board-certified radiologists among the authors . In total, 27 articles met our eligibility criteria Table . These r“Extra-axial variant of medulloblastoma” is a descriptive term indicating that the lesion originates externally to the brain parenchyma and not from an exophytic outgrowth, beyond the pial surface  . Based oThe CPA is an extra-axial wedge-shaped cisternal space bounded by the lateral petro-temporal bone, cerebellum, and brain stem medially, as well as by the inferior cranial nerves  . Most poThere are various clinical presentations of CPA medulloblastomas, which are difficult to distinguish from those of other CPA neoplastic or non-neoplastic lesions . InteresThe primary components of CPA medulloblastoma are two molecular subgroups, including WNT-activated and SHH-activated medulloblastoma . CPA medThe cerebellar tentorium is a crescent-shaped dural reflection that extends across the posterior cranial fossa, separating the occipital and temporal brain hemisphere from the cerebellum and the infratentorial brainstem .The tentorial location is considered to be the second most common location of medulloblastomas after the CPA. The presence of medulloblastomas at this location was first described by Becker et al.  in 1995.The lateral cerebellar region, which includes the retro-cerebellar region, is considered the third most common location after the CPA and tentorial regions. This site is bounded beyond the border of the lateral cerebellar hemisphere and also includes the retro-cerebellar site and is marginated by the mastoid part of the temporal and occipital bones. It is an extremely rare location for an extra-axial medulloblastoma. Medulloblastoma cases in this location were first reported by Meshkini et al.  in 2014.Medulloblastomas in this location are assumed to arise from the remnants of the external granular layer in the cerebellar hemisphere. Similar to medulloblastomas at the tentorial location, it is difficult to differentiate medulloblastomas in this region from meningiomas or other pathologies, especially when medulloblastomas exhibit dural tail signs, which is usually observed in meningiomas in this or in the tentorial location Table . The latThe foramen magnum is the largest foramen of the skull, which is located in the most inferior part of the cranial pit as a part of the occipital bone . The forRecently, there have been tremendous advances in diagnostic neuro-oncology. Accordingly, it is currently possible to detect this devastating tumor at an earlier stage. For example, in vivo detection of the receptor status using positron emission tomography (PET) scan on mothers has enabled diagnosticians to anticipate the potential consequences.Although at present, radionuclide imaging is not the primary diagnostic modality for intracranial tumors, primarily limited by its low specificity and low spatial resolution. In 1998, Müller et al.  evaluateRadionuclide imaging has a limited potential in identifying medulloblastoma from differentiated diagnoses. However, the fusion of CT or MRI images with PET images can improve PET’s ability to diagnose and distinguish post-radiotherapy changes from tumor recurrence . This isRadionuclide imaging shows a limited potential capability in diagnoses and identification of medulloblastomas compared with differential diagnoses, and medulloblastomas have a limited potential for uptake in thallium-201 single-photon emission CT and fluorodeoxyglucose PET  .Dynamic perfusion parameters have recently been evaluated for their potential diagnostic roles in neuro-oncology, especially for lesions of predominantly nonnecrotic solid tumors , 40, 41.Dynamic susceptibility contrast imaging of medulloblastomas has revealed increased permeability, with the cerebral blood volume ratios close to 1 , particuMedulloblastomas are considered as tumors with the greatest relative tumor blood flow Fig. , which cFinally, it is noteworthy that the characteristic arterial spin-labeling perfusion patterns have been studied among diverse pathologic types of brain tumors in children. These studies have revealed that the maximum relative tumor blood flow of high-grade tumors (grades III and IV) is significantly higher than that of low-grade tumors (grades I and II) , 41.Quantitative Apparent Diffusion Coefficient (ADC) value analysis can facilitate preoperative identification of medulloblastoma from its differentials, as well as grading of pediatric medulloblastoma , 43. FurThe ADC ratio—the proportion between the mean ADC observed in the tumor and the mean ADC observed in the contralateral white matter—is a simple tool used to distinguish juvenile pilocytic astrocytomas, ependymomas, and medulloblastomas . In partMRS based on the metabolic pattern can be used to identify medulloblastomas Figs. C, 7C. A MR perfusion is a distinguishing modality for posterior fossa diagnosis. In 2014, Yeom et al.  clarifieClinical assessment and neuroimaging findings are insufficient in obtaining an accurate preoperative diagnosis of extra-axial medulloblastomas because of shared features between medulloblastomas and other common pathologies. Most reported cases of posterior fossa extra-axial medulloblastomas are located in the CPA. Radionuclide and arterial spin-labeling imaging are newer and significantly useful techniques for the diagnosis and recurrence detection of medulloblastomas. Moreover, the somatostatin receptor subtype 2 might be a potential prognostic marker and therapeutic target for medulloblastomas. We believe that cases of medulloblastomas are likely under-reported because of publication bias and a tendency to report usual tumors at unusual locations. The information provided in this review would help establish a more accurate understanding of these lesions as one of the essential roles of a neuroradiologist is to detect the tumor and possible subarachnoid spread, which determines and guides the surgical approach.


Tremor is a hyperkinetic movement disorder most commonly encountered in essential tremor (ET) and Parkinson’s disease (PD). The purpose of this review is to summarize molecular neuroimaging studies with major implications on pathophysiological and clinical features of tremor.Oscillatory brain activity responsible for tremor manifestation is thought to originate in a cerebello-thalamo-cortical network. Molecular neuroimaging has helped clarify metabolic aspects and neurotransmitter influences on the main tremor network. In ET, recent positron emission tomography (PET) studies are built on previous knowledge and highlighted the possibility of investigating metabolic brain changes after treatments, in the attempt to establish therapeutic biomarkers. In PD, molecular neuroimaging has advanced the knowledge of non-dopaminergic determinants of tremor, providing insights into serotonergic and noradrenergic contributions.Recent advances have greatly extended the knowledge of tremor pathophysiology and it is now necessary to translate such knowledge in more efficacious treatments for this symptom. Tremor is an involuntary, rhythmic, oscillatory movement of a body part. According to the latest consensus statement on the classification of tremor of the International Parkinson and Movement Disorder Society, tremor may be classified along two axes, i.e. clinical features axis 1) and etiology axis 2) .–27.19–2711C-flumazenil, a GABAA receptor ligand, PET was used in one study to assess the state of the GABAergic system in 8 ET patients and 11 controls. Increased tracer binding, corresponding to an increased GABAA receptor availability, was found in the ventrolateral thalamus in the ventral intermediate (Vim) nucleus, dentate nucleus and premotor cortex, sites of the tremor network. The increased GABAA receptor availability is hypothesized to be a receptor upregulation process caused by a deficit in GABAergic transmission that could be due to either GABA deficiency or to the presence of abnormal and dysfunctional GABAA receptor subtypes [11C-flumazenil PET study in 10 ET patients found a positive correlation between cerebellar tracer binding and tremor severity scores [A few studies have provided investigations about the state of other neurotransmitter systems in ET, such as the GABAergic and the serotonergic systems. Reduced GABAergic function in the tremor network is thought to play an important role in ET and is likely the basis of its improvement with GABAergic medication. Pathological studies have shown cerebellar degenerative changes, including a decrease in the number of GABAergic Purkinje cells . 11C-flusubtypes . Anothery scores . GABA coy scores . OverallFinally, only two small imaging studies have investigated the state of the raphe serotonergic nuclei through SERT tracers, and they did not highlight changes compared to controls or correlation to tremor scores , 38.PD is characterized by rest, postural and kinetic tremors; a subtype of postural tremor, re-emergent tremor, may also be present , 39.Rest tremor is the most common type of tremor in PD and thought to be generated by abnormal activity in the basal ganglia that induces tremulous activity in a cerebello-thalamo-cortical tremor network . More rePerfusion abnormalities studies were among the first to investigate PD tremor. These small studies in patients with deep brain stimulation (DBS) of the ventral intermediate nucleus (Vim) of the thalamus found cerebellar blood flow increases during tremulous activity that was reversible upon successful DBS activation and subsequent tremor arrest –45. Inte18FDOPA PET showed that higher dopaminergic terminal availability in the striatum of tremor-dominant patients was also associated with a higher glucose metabolic rate compared to akinetic-rigid patients [The study of cerebral metabolism through FDG-PET has revealed interesting findings in PD tremulous phenotypes. PD shows a classically altered metabolic signature, named the PD-related pattern (PDRP), characterized by increased pallidothalamic and ponto-cerebellar metabolism and relative reductions in premotor and posterior parietal (associative) cortical regions –48. Notapatients . OverallNeurotransmitter dysfunction has been extensively investigated in PD tremor especially through dopaminergic imaging. Nigral degeneration and associated striatal dopaminergic loss are the hallmark of PD and thought to be necessary for the generation of PD tremor. However, as highlighted in the previous paragraph, phenotypic expression of rest tremor may be sometimes unrelated to nigrostriatal degeneration, as it is in some cases of essential tremor with rest tremor. A further complication in establishing the pathophysiology of PD tremor is the fact that its clinical severity seems unrelated to the degree of nigrostriatal denervation, as opposed to bradykinesia and rigidity –61. In o1A) ligand uptake in the midbrain raphe correlated inversely with total and rest tremor scores, but not with bradykinesia and rigidity [123I-FP-CIT SPECT, showed inverse correlations with tremor severity in the entire cohort of PD patients and in the tremulous subgroup [The lack of correlation between nigrostriatal denervation and rest tremor severity, coupled with mixed responses to dopaminergic medication, has sparked research into non-dopaminergic determinants of PD tremor, especially serotonin and noradrenaline. A first PET report on the serotonergic influence on tremor showed that a serotonin receptor , a selective NET ligand, have highlighted different aspects of noradrenergic dysfunction in PD. It was shown that tremulous patients had significantly higher tracer uptake in the locus coeruleus and thalamus compared to non-tremulous patients. Tremulous patients had a locus coeruleus uptake similar to controls whereas the raphe was the only site with a significant reduction. Furthermore, rest tremor scores were positively associated with median raphe, thalamus and red nucleus tracer uptake [Noradrenergic dysfunction has been shown to influence tremor in PD. Central noradrenergic projections originate from the pontine locus coeruleus, known to be activated under stressful conditions. These are known in turn to exacerbate tremor . Moleculd tremor . More rer uptake , 76•. LiOverall, molecular neuroimaging studies investigating PD tremor have helped clarify many pathophysiological aspects. One caveat in interpreting some of these studies is that tremulous phenotypes, rather than tremor itself, were investigated. Therefore, while many studies did establish associations with tremor severity, thus likely indicating a direct influence on tremor itself, others described associations with a specific phenotype. The latter must be interpreted with caution in terms of tremor pathophysiology, since abnormalities may be due to the phenotype itself, which may in turn be due a specific neuropathological pattern, rather than being directly associated with tremor.Tremor may be a clinical feature associated with dystonia, and dystonia often presents with tremor. Furthermore, isolated tremor syndromes such as isolated head tremor may be forms of dystonia without clear abnormal posturing –79. A reHolmes tremor is a syndrome of low frequency (< 5 Hz) rest, postural and kinetic tremor that involves proximal and distal muscles . It is gA receptor abnormalities have also been found and shown to be associated with tremor severity [Tremor is a common hyperkinetic movement disorder with a complex pathophysiology. The most frequent tremor causes are essential tremor and Parkinson’s disease tremor. A shared pathophysiological substrate of tremor likely lies in a cerebello-thalamo-cortical network capable of generating tremor oscillations and ultimately the involuntary movements. Neurotransmitter systems outside this network, such as dopamine, noradrenaline and serotonin, are able to influence the clinical manifestation of tremor. Molecular neuroimaging has provided great insights into the clinical correlates of tremor in ET and PD. In ET, cerebral blood flow and metabolic abnormalities have been characterized and they have demonstrated potential for translation into useful therapeutic biomarkers •, 17•. Gseverity , 36. ConRest tremor is one of the hallmarks of PD and it is thought to arise from basal ganglia dopaminergic dysfunction and its influence on the cerebello-thalamo-cortical network, a dysfunctional system that is thought to work in a “switch and dimmer” fashion . The degOver the past few years, the pathophysiology of tremor has become increasingly clearer. However, both in ET and PD available medications may not be efficacious in all patients.Therefore, there is a great need to transform an increasing wealth of knowledge about the mechanisms of tremor into new treatments.


The risk of infection-related morbidities and mortality is particularly high in the newborn period. Before birth, the mammalian fetus is protected from adverse effects of exogenous pathogenic microbes and can normally develop its immune system in a near-sterile environment with limited need for immune responses. This condition changes dramatically at birth when rapid adaptations of the innate and adaptive immune systems are required to tolerate and respond to commensal and pathogenic bacteria at epithelial surfaces  and fight microbes penetrating to blood and internal organs. Carefully balanced responses of the systemic, organ-related and epithelial immune systems are required to avoid bacterial overgrowth, translocation across immature barriers, and excessive inflammation. The many arms of the mammalian immune system develop differently in different species but comparative studies facilitate insights into mechanisms of perinatal immune development and help identify prophylactic and therapeutic opportunities. Interventions to support neonatal immunity are most critical for those born preterm, growth-restricted, hypoxic, infected or otherwise compromised at birth.www.neomune.ku.dk, 2013-20), having a focus on milk and microbiota influences on gut, immunity and brain development. The Research Topic and this editorial combine knowledge obtained in the NEOMUNE consortium with a series of complementary articles. We encourage studies into the mechanisms of systemic and mucosal immune development, and how dietary, microbial and pharmacological interventions support immune maturation assessed by both classical immune markers and exploratory omics techniques. The latter methods have recently emerged as novel tools to better understand immune development and prepare the way for a new precision medicine approach to the prevention, diagnosis and treatment of neonatal immune disorders  and maternal immunity protect the developing fetus against infections. After birth, the protective and immunomodulatory properties of colostrum and milk provide continued support, concomitant with a gradual development of both the innate and adaptive arms of the newborn immune system. The innate immune system and the epithelial barriers are the first line of defense against infections and immune cells can react rapidly, non-specifically and pre-programmed to combat infectious stimuli before more adaptive immunity develops. The cellular, structural and functional elements of the immune system may remain distinct from those in older individuals for days, weeks or months after birth, yet this special early life immune status may also confer certain survival benefits for the host. Thus, a relatively ‘dormant’ immune system may support a physiologic and metabolic state that helps to dampen hyper-inflammatory responses following the sudden exposure of the newborn host to a world of microbes , 4.What then if newborns are born too early and/or too small? Annually, an estimated 20 million infants  are born preterm  and/or growth-restricted (< 10% growth percentile). Their health complications account for up to half of all infant deaths , 6. PretIn humans, spontaneous preterm birth is related to one of two overlapping disease etiologies: Fetal infection/inflammation, leading to placental dysfunction, or placental vascular dysfunction, causing hypertensive disorders and fetal growth restriction (with/without preterm birth). Dramatic advances in neonatal medicine, including ventilator management, nutrition and medical therapies now allow good survival rates of extremely preterm infants from 60% GA . Yet, the immature state of many organs at birth predisposes to later complications, many of which have a clear immunological or inflammatory component . Understanding the complexity of all these interacting diseases in early life of preterm infants demands a multi-organ research approach and appropriate animal models of immune-related disease in early life.Viemann reviewed how alarmins, e.g., S100A8/9, are crucial factors for perinatal immune regulation. Based on a large cohort of Gambian children, Odumade et al. indicated that developmental changes in adenosine deaminase (ADA) levels may also play a role for this immunological transition. In therapeutic settings, adjunct therapies to antibiotics, such as the phosphodiestarease inhibitor pentoxifylline (PTX), may help to dampen damaging pro-inflammatory responses, without limiting bacterial clearance, as shown by Speer et al. in newborn mice infected with Gram-negative E. coli. Similar anti-inflammatory effects in newborns are shown after Gram-positive infection of newborn mice  collections from preterm piglets can be used to reveal new proteome biomarkers of neonatal Gram-positive infection, potentially affecting both systemic and brain compartments, and how responses are modulated by milk diets.Successful perinatal immune adaptation is a delicate balance between tolerance and resistance of the immune system to pathogens and/or inflammatory factors, with bacterial colonization along host epithelial surfaces and diet playing important roles. Many endogenous host factors may support the development of balanced immune responses. In this Research Topic, Sampah and Hackam and Sproat et al. add important information to existing reviews on immune ontogeny in infants and include description of the various cellular components . Across preterm infants, pigs and newborn mice, there is up-regulation of circulating cell-free DNA and neutrophil-associated proteins at or shortly before LOS and/or NEC  and specialized cell types support immune tolerance and sensitivity to inflammatory insults. The review by  studies  and bloo studies , 18.Westrøm et al. provides an updated review on the knowledge of the immature gut barrier and the transfer of immunoglobulins from mother to young across species, and its role in immune maturation. Such passive immunoglobulin transfer may have effects beyond immune protection in gut and blood, potentially affecting more distant organs such as the immature brain, as reviewed by Pierzynowska et al.Systemic immunoglobulin G produced by B cells is the most abundant antibody type in the body, binds to cell surface receptors on many cell types to stimulate immune cell phagocytosis, cytotoxicity and activates complement cascades. Thus, systemic transfer of immunoglobulins from mother to offspring during the fetal and/or neonatal period helps to expose naïve immune cells to antigens in a controlled fashion, regulate their pathogen responses and stimulate maturation of the immune system, facilitate tolerogenic responses and prevent excessive inflammatory responses. Distinct differences exist among mammals in the time and mode of transmission of this passive immunity. In newborn ungulates  this occurs mainly via uptake of colostrum via the gut while for infants this occurs in utero across the placenta in the last third of pregnancy. The Research Topic review by Bæk et al. In preterm newborns, such infections may lead to inflammatory lesions in various organs, including the immature brain , may cause serious organ injury, including in the CNS. The tissue- and age-specific interaction between CMV and host cells depends critically on macrophages and monocytes, facilitating the balance between ‘longer-term immune benefits or immunopathology´, as elegantly reviewed by Baasch et al. in this Research Topic. Possibly, diet-dependent effects of the developing gut virome and bacteriome determine later-life mucosal immunity via cell-specific epigenetic changes . Consistent with this, brain inflammatory responses were also demonstrated after development of severe NEC lesions in preterm pigs  pathways. This helps to explain the commonly observed association between gut bacterial dysbiosis and NEC, growth restriction and later lung BPD development in preterm infants.Locally in the gut, toll-like receptors are important to recognize pathogen-associated molecular patterns (PAMPs) in early life and could trigger innate immune responses not only in the gut, but also in distant organs, such as the lung and brain, by signaling the recruitment of immune cells to these organs and facilitate secondary immune responses, as reviewed by al cells , or correrm pigs , 27. In Villamor-Martinez et al. shows that the overall moderate CA-associated increase in later infections  partly relates to CA infants being born at lower GA while the CA effect on infant infections at birth is clear . By transcriptomic analyses on cord blood, Golubinskaya et al. showed high expression of S100A alarmins in cord blood monocytes of CA-exposed preterm infants, potentially helping to diagnose and treat CA effects after birth. Such associations show the need to do well-controlled studies in animals, allowing separation of the effects of preterm birth from its fetal and maternal predisposing factors, and thereby understand mechanisms across species and organs.Inter-organ immune communication and inflammatory insults may occur even before birth, preterm or term, and affect postnatal immune development. Chorioamnionitis  is a key predisposing factor for preterm birth and adverse postnatal outcomes in CA infants  are well known but responses are highly time-, GA-, age-, organ- and pathogen-specific. There are reports of reduced LOS risk following CA in preterm infants , and fetHeymans et al. demonstrated in cohorts of fetal lambs that fetal inflammation induces marked structural changes to the immature gut, including its enteric system and immune-regulatory glial cells. From lamb studies, it remains unclear if such effects predispose to later development of NEC lesions because preterm lambs are difficult to rear postnatally. In preterm pigs, few days of intra-amniotic exposure with LPS (reflecting Gram-negative infection) had limited or no effects on later NEC sensitivity , with differential effects on immune development. Independently of predisposing factors, IUGR infants show increased infection susceptibility, probably due to low blood leukocyte, lymphocyte and macrophage counts just after birth, while specific effects on gut and lung inflammatory disorders  are more limited , 34. Yetnewborns , 13, 36.Gravina et al. demonstrated in mice that the brain-damaging effects of hypoxia were most pronounced in male pups and during the acute phase of systemic infection with gram-positive bacteria. Male-specific higher mortality and immune defects were also demonstrated in the immediate postnatal period of preterm pigs  interventions may have most pronounced effect on immune development in the first days and weeks of life, especially when birth occurs preterm. Initially, these infants are often nourished parenterally but lack of luminal gut stimulation may in itself lead to impaired mucosal and systemic immune development via deficient gut trophic and immunomodulatory effects  and disturbed bacterial colonization. Further support for early enteral nutrition is related to the risk that excessive supply of parenteral metabolic substrates  may create hyperinflammatory responses in immature immune cells .Sproat et al. summarized the factors in milk  that may affect innate and adaptive immunity in immature newborns. The first milk, colostrum, is particularly rich in immune-regulating factors and these may act to enhance immune defense in both species-specific and species-unspecific ways. Recently, bovine colostrum was investigated as a supplementary diet for mother´s own milk, formula or donor human milk for preterm infants . Further, Pan et al. demonstrated epigenetic programming and NEC-protective effects of early colostrum supplementation on gut mucosal immunity and gene expression in newborn preterm pigs on parenteral nutrition  on the developing immune system via changes to the gut microbiota are demonstrated across many studies. However, due to wide individual variation in gut microbiota composition, type of pre- and probiotic products, and their interactions with milk diet and the non-bacterial gut microbiota , many questions remain unresolved. Across trials and products tested, dietary pre- and probiotics reduce NEC, and to a lesser degree, LOS in preterm infants , 54. IncOosterloo et al. in this Research Topic shows that immune-related markers in plasma of term infants may be affected up to 1 year after neonatal exposure to AB. In piglets, Hu et al. showed that low-dose, longer-term AB treatment reduced Th1-related blood immune responses while intestinal innate immunity-related genes were enriched, together with reduced IFN‐γ and IL‐6 expression. Together these studies indicate that AB-treated hospitalized infants could be more sensitive to later gut and systemic infections, especially if treated for longer periods  had limited effects. On the other hand, neonatal AB treatment by the oral route seemed to delay gut colonization, improve gut immune gene expressions, plasma proteins and metabolites, indicating immune maturation and reduced systemic inflammation , together with completely new modes of nutrition, respiration, metabolism and excretion. Via 29 reviews and original research articles this Research Topic has shown how perinatal immunity develops in compromised newborns. Dysregulated immune development in newborns born preterm, growth-restricted, infected or subjected to placental dysfunction or birth asphyxia - is not a surprise. Rather, it is surprising how well these infants survive, adapt and thrive, despite their increased susceptibility to infections and inflammation in early life. Due to the age-, cell–, organ- and species-specificity of the developing immune systems, only a few interventions enhancing bacterial protection and immunity have become universally accepted for sensitive infants . Novel dietary, microbiota and pharmacological interventions still require better documentation and evidence of mechanisms. Cross-species studies and omics-based analyses may help to understand mechanisms and ensure healthy development without side effects. Newborns that are compromised by fetal growth restriction, inflammation, infection, preterm birth and/or delivery complications often experience immunological deficits in the immediate postnatal period. Yet, their adaptive capacity is remarkable, making immune modulation of compromised newborns a difficult balance between potential benefits and possible harm.PS, TS, AC and DN served as Guest Editors in the Research Topic. PS wrote the outline of this manuscript. TS, DN and AC reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.The work was funded in part by grants from Innovation Fund Denmark (NEOMUNE and NEOCOL grants).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Natural polymers, based on proteins or polysaccharides, have attracted increasing interest in recent years due to their broad potential uses in biomedicine. The chemical stability, structural versatility, biocompatibility and high availability of these materials lend them to diverse applications in areas such as tissue engineering, drug delivery and wound healing. Biomaterials purified from animal or plant sources have also been engineered to improve their structural properties or promote interactions with surrounding cells and tissues for improved in vivo performance, leading to novel applications as implantable devices, in controlled drug release and as surface coatings. This review describes biomaterials derived from and inspired by natural proteins and polysaccharides and highlights their promise across diverse biomedical fields. We outline current therapeutic applications of these nature-based materials and consider expected future developments in identifying and utilising innovative biomaterials in new biomedical applications. Traditional biomaterials used in biomedicine, such as gelatine, silk and collagen, were derived from natural sources , with thBiologically inert materials were originally favoured for biomedical applications on the basis of safety and stability. Years of clinical use have identified that even inert mate-rials may elicit damaging cellular and immunological responses, however ,7. As a This review describes the broad range of current biomaterials that are derived from, or inspired by, natural proteins and polysaccharides. We summarise the diverse biomedical and biotechnological roles successes of these materials to date, in fields such as regenerative medicine and therapeutics. Finally, we consider potential future directions for the development and modification of novel biomaterials with broader and more effective biomedical applications.Choosing a suitable biomaterial for, e.g., scaffold construction is critical for its success. In vivo target sites at which biomaterials are used differ greatly and include soft   and hardBiodegradable polymeric biomaterials can be divided into natural and synthetic materials, based on their origin and whether they contain naturally occurring extracellular matrix (ECM) . CommonlScaffolds that act as templates for tissue regeneration and guide the development of new tissues can be produced from a diverse range of materials  18]. Na. Na18]. Scaffolds should allow or promote cells to adhere, proliferate and spread before creating a new matrix, and must not elicit an inflammatory response that could lead to infection, longer healing times or patient discomfort .Scaffolds should ideally be temporary templates which will be replaced by newly regenerated tissue . TherefoScaffolds must be highly porous to promote cell migration, waste dispersal, scaffold-tissue interaction and nutrient and fluid permeability . Cell-biScaffolds must have sufficient mechanical strength to maintain their structural integrity—including during transport, surgical handling and implantation ,28,29. EScaffolds must be cost-effective to produce and easily scalable from laboratory production. Production must also be compatible with good manufacturing practice (GMP) standards  for tranIn nature, an array of proteins play vital structural roles in living organisms, which has led to their incorporation in recent years into protein/polypeptide-based biomaterials based on their structural chemistry, cellular interactions or cell communication properties . Non-strCollagen is the most abundant structural protein in humans and animals. It makes up approximately 30% of all mammalian proteins and is an essential component of the ECM . By virtThe collagen family consists of 29 distinct collagen types which are divided into four classes based on their composition and structural properties . All typCollagen is weakly immunogenic, with fibrillose collagen exhibiting lower immunogenicity than smaller molecules due to the burial of potentially antigenic sites during its auto-polymerisation . RemovalSpecific peptide sequences in collagen bind four different types of cell-surface receptor. GPO (Gly-X-Y) motifs, where X and Y can be any amino acid residue but are most commonly proline , bind tyCollagen can be obtained for biomedical applications from mammalian sources such as cows, pigs, rats and sheep ,115, as Extraction and purification of collagen from natural sources can also be carried out by solubilisation and purification. Due to its covalent cross-linking, collagen exhibits low solubility in organic solvents and is typically solubilised in acidic (0.5 M acetic acid), neutral salt (0.15–0.20 M NaCl) or proteolytic solutions.Unlike decellularised collagen which has been naturally polymerised in vivo, extracted collagen must be cross-linked to increase its mechanical strength and resistance to enzymatic degradation. There are several well-established methods of cross-linking, including physical processes such as ultra-violet (UV) or thermal treatment , via cheWhether purified via decellularisation or solubilisation, extraction and cross-linking, collagen must be sterilised for in vivo use. Due to its relatively fragile, temperature-sensitive structure, sterilisation methods can alter its molecular properties. Even low-dose gamma irradiation reduces its enzymatic resistance and mechanical strength, though the addition of glucose can mitigate these effects by mediating cross-linking . β- and As collagen is primarily obtained from animal sources, the transmission of infectious disease is a concern in therapeutic applications. The recent COVID-19 pandemic exemplifies the dangers of zoonotic infections, while cases of bovine spongiform encephalopathy have resulted from the use of prion-contaminated bovine scaffolds . MeanwhiEscherichia coli is the best-established expression system for recombinant proteins due to its ease of genetic manipulation, rapid growth rate, track record in protein production and suitability to scale-up .,69.68,69Unlike most applications of nanocelluloses, BNC-based biomaterials have moved past clinical trials and into common clinical practice. Their primary use is in the treatment of non-healing lower extremity ulcerations, where the use of BNC biomaterials has been reported to reduce healing times from a mean of 240–390 days to 80 days . Many BNCMC and cellulose nanocrystals have also been incorporated into numerous bioinks to improve their viscosity for printing and to provide mechanical strength to printed hydrogels . This haCellulose and nanocellulose clearly have widespread applications across wound healing, drug delivery and as substitute implants, due largely to their biocompatibility, versatility and tuneable chemical and physical characteristics. Their inherent lack of degradability when used as scaffolds may be the main limitation of nanocellulose biomaterials, so that their greatest promise may reside in drug delivery and wound healing, especially when combined with antimicrobial agents for improved effectiveness.N-acetyl glucosamine residues of chitin are completely deacetylated, giving rise to N-glucosamine [N-acetyl D-glucosamine) and (1-4)-2-amino-2-deoxy-β-D-glucan (D-glucosamine) units  units  253]. T. TN-acetChitosan is produced by deacetylation of its parent molecule chitin. While typically carried out using concentrated sodium hydroxide and sodium borohydride to prevent reactive species generation and chitin depolymerisation , numerouThough a polysaccharide, chitosan exhibits multiple amino groups which render it degradable by human proteases such as lysozyme in vivo . In addiChitosan is well known for its antimicrobial and antifungal properties, which make it attractive for use in biomedical scaffolds. The basis for its antimicrobial activity is uncertain but its positive charge is believed to interact with the negatively charged cell surfaces of microorganisms and inhibit both uptake and excretion of materials . Low molRecent in vitro and in vivo studies have highlighted the antitumor activity of chitosan, which is thought to result from an increase in interleukin (IL)-1 and IL-2 secretion due to the infiltration of mature cytotoxic T lymphocytes ,268. ChiChitosan can aid blood clotting and reduce pain via blocking of nerve endings . These hThe biodegradability and antimicrobial, anti-tumour, haemostatic and mucoadhesive properties of chitosan have led to it finding numerous applications in biomedicine and pharmaceuticals, as outlined below.E. coli in vitro and in vivo . Th. Th85]. Azotobacter species, extremely rigid due to very high concentrations of G-blocks [G/M ratios, length of G blocks and the sequence pattern of GM block repeats all affect the physical properties of alginate hydrogels . AlginatG-blocks , which iDespite extensive research on the biocompatibility of alginates, the effect of the molecular structure on biocompatibility remains uncertain. While alginates high in M blocks have been reported to be highly immunogenic and to stimulate cytokine production at 10-fold higher levels than alginates with a high G block content , other iAlginate is not degraded by mammals as they lack the alginase enzyme responsible for cleaving the polymer chains . AdditioMost commercially available alginates are derived from the above kelp species, typically harvested from natural populations as cultivation is considered too expensive for industrial production. While alginate biopolymers can make up almost 40% of the dry weight of the brown seaweeds , their qWhile other biomaterials are utilised in many different formats, such as sponges, films and microparticles, alginate is primarily used in the form of hydrogels. These hydrogels can be formed by a variety of gelling mechanisms to modify their functionality.2+ cations, typically in the form of calcium chloride [Alginate hydrogels are most commonly prepared by the addition of ionic cross-linking agents such as divalent Cachloride . The catchloride . Bufferschloride ,292. As chloride .2+ ions released via this process can promote haemostasis, while the resultant poorly cross-linked gel may also lead to aggregation of red blood cells and platelets [Stability issues may arise when gels formed through ionic cross-linking are incubated under physiological conditions for extended periods of time, however, due to exchange reactions with surrounding monovalent cations . Ca2+ iolatelets . TherefoCross-linking of alginates using PEG amines of different molecular weights has been investigated to produce gels with a broader range of mechanical properties. Increasing the cross-linking density/amount (weight) of PEG led to improved mechanical strength and increased Young’s moduli in gels ,295. ConAs mammals lack the enzymatic capacity to break down alginate chains, this jeopardises the potential biomedical usefulness of alginates, as biodegradation and safe excretion from the body are primary concerns for biomaterials in vivo . While iMultiple approaches have been developed to produce biodegradable alginates for in vivo applications. Partial oxidation of alginate leads to increased degradability in aqueous environments  due to cAs mammals lack cell receptors for alginate polymers, coupling of molecules such as fibronectin  and collAlginate gels have been widely applied across various fields of biomedicine, including wound healing, tissue/bone regeneration and as model systems to study mammalian cell culture .Ionically cross-linked alginate gels can be processed by freeze-drying to produce foam-like porous sheets and fibrous dressings which provide a moist environment more suited to wound healing than traditional dressings . The dre2+ cross-linked outer gel matrix to act as a delivery vehicle.Alginate gels have been used for sustained delivery of both low molecular weight chemical and protein drugs. The former diffuse rapidly from nanoporous (pore size ~5 nm)  alginateAlginate has also proven an excellent delivery vehicle for protein-based drugs as proteins are easily incorporated into alginate gels without denaturation and the gels protect the proteins from degradation and allow their controlled release. Protein release rates are typically fast due to the gels’ hydrophilic properties and porous structures , though B. mori silk fibroin over alginate microspheres has been utilised to achieve a mechanically rigid structure which protects enclosed proteins and prevents their diffusion [The fast release of most protein drugs from alginate gels can be slowed by gel cross-linking techniques which can increase the incidence and strength of protein-gel interactions . Insuliniffusion .Alginate gels have been widely utilised to transport proteins and cells for regeneration or engineering of tissues and organs in the body. As molecules smaller than 5 nm are quickly released from the porous alginate gel whereas those larger than the pore size are only released following gel dissociation, plasmid DNA molecules of approximately 100 nm in diameter are released from alginate gels only upon degradation .Alginate gels have been used to stimulate angiogenesis and partly relieve ischemia in murine tissue through sustained and localised release of growth factors such as VEGF . Gels haAlginate gels are also attractive in bone regeneration as they can fill irregular shapes that other scaffolds may not and can be introduced into the body non-invasively and easily modified to carry cell-adhesion ligands or growth factors. Their inability to bear weight is of some concern  but theyAlginate is also one of the most commonly used biomolecules in 3D bioprinting due to its compatibility with extrusion and inkjet printing processes , and itsAlginate has shown considerable potential as a biomaterial, particularly in wound healing, guided tissue engineering and drug delivery. While in hydrogel form, it exhibits very promising gelling capability, ease of modification and biocompatibility, the poor mechanical strength of other alginate forms limits their potential applications. Future research is likely to see the development of dressings with much higher bioactivities, as well as modified carrier vehicles for drug and bioactive molecules. Functionalisation with a wider range of molecules and engineering the molecules’ physical and chemical properties will be key to developing application-specific, tissue-interactive alginate biomaterials.Natural polymers, both protein- and polysaccharide-based, display enormous potential in tissue engineering, drug delivery and diverse biomedical applications. Traditional materials previously designed to be inert in order to be “biologically safe” no longer meet the needs of modern medicine, where biomaterials are designed to make extensive biological and physical interactions with tissues and cells for improved in vivo performance. The ECM-like characteristics of natural polymers, their similarities to many human proteins and their natural versatility have led to the modification of well-established biomaterials, as well as the development of new materials, that have found novel applications in tissue engineering, as implantable structures and as vehicles for controlled drug release.Though enormous progress has been made in the field of nature-based biomaterials in recent decades, room for improvement remains. Novel biomaterials which outperform current state-of-the-art offerings in terms of reduced side effects, lower purification or manufacturing costs and greater therapeutic efficacies will continue to be developed. Researchers continue to search for new natural sources of biomaterials which, together with innovative scaffold fabrication techniques, will lead to more effective biomaterials and new applications. The recent successes with natural materials and the broad array of materials currently available will inspire researchers to continue to look to nature for novel biomaterials, however, with fundamental biological and chemical understanding of such materials remaining central to the emergence of new, innovative biomaterials for new biomedical applications.


Severe spinal cord injury causes permanent loss of function and sensation throughout the body. The trauma causes a multifaceted torrent of pathophysiological processes which ultimately act to form a complex structure, permanently remodeling the cellular architecture and extracellular matrix. This structure is traditionally termed the glial/fibrotic scar. Similar cellular formations occur following stroke, infection, and neurodegenerative diseases of the central nervous system (CNS) signifying their fundamental importance to preservation of function. It is increasingly recognized that the scar performs multiple roles affecting recovery following traumatic injury. Innovative research into the properties of this structure is imperative to the development of treatment strategies to recover motor function and sensation following CNS trauma. In this review, we summarize how the regeneration potential of the CNS alters across phyla and age through formation of scar-like structures. We describe how new insights from next-generation sequencing technologies have yielded a more complex portrait of the molecular mechanisms governing the astrocyte, microglial, and neuronal responses to injury and development, especially of the glial component of the scar. Finally, we discuss possible combinatorial therapeutic approaches centering on scar modulation to restore function after severe CNS injury. The failure of repair following spinal cord injury (SCI) is due to factors both intrinsic to cellular components and extrinsic, surrounding the site of injury. In the 1980s, the Aguayo lab first showed that long-distance axon regeneration from CNS neurons within a peripheral nerve graft was abruptly halted at the border between the Schwann cell-laden peripheral nervous system (PNS) and astrocyte-rich central nervous system (CNS) , and how alleviating these glycoproteins in combination with other strategies can help promote functional recovery.Lineus sanguineus  are able to completely regenerate the anterior aspect of their bodies meaning 200,000 worms could be generated following a similar number of dissections, each just 1/200,000th the volume of the original animal   which is born precociously. Up to 12 days after birth, cervical segments of the opossum cord do not scar  which signal via a newly appreciated relationship with autophagic flux  injury to the spinal cord, which immediately causes axon shearing, bleeding, and cell death. This leads to the release of alarmins, which recruit local microglia and systemically circulating immune cells that pass through the damaged blood-spinal cord barrier to propagate inflammation and secondary damage  the lesion site , characterized after being activated by zymosan, were found to drive retinal ganglion cell axon regeneration by secreting growth factors such as NGF and IGF-1 , which aids embryo survival and selective autophagy  and chronic (8 weeks) post-SCI glial scar , which surround the soma and proximal dendrites of certain subpopulations of neurons including fast-spiking parvalbumin and GABAergic neurons   macrophages, microglia, oligodendrocyte progenitor cells, pericytes/fibroblasts, ependymal cells, and endothelial cells, with neuron growth cones entrapped in the lesion penumbra. Here, we have discussed how next-generation sequencing technologies have revealed how complicated this post-injury structure is at the cellular, molecular, and transcriptomic levels with an emphasis on the pre- and post-astrocytic, microglial, and neuronal cellular heterogeneity. Acutely, this structure is indispensable to the wound healing process to limit further secondary tissue damage and to resolve rampant inflammatory processes. Chronically, however, the typical glial/fibrotic scar that forms after contusive or extensive surgical lesions with a balance of extracellular matrix proteins tipped in the direction of inhibition, including CSPGs, poses a hugely inhibitory biochemical and physical barrier to successful axon regeneration and functional recovery. Our understanding of the uniquely heterogeneous composition of the scar and its evolution over time advocates for a combinatorial strategy to encourage axon regeneration and functional recovery. Treatment strategies that simultaneously target both the intrinsic problem of decreased axon outgrowth machinery as well as the extrinsic hurdles of increased axon-inhibitory proteins coupled with targeted rehabilitation are our best options going forward.


The management of retinal diseases relies heavily on digital imaging data, including optical coherence tomography (OCT) and fluorescein angiography (FA). Targeted feature extraction and the objective quantification of features provide important opportunities in biomarker discovery, disease burden assessment, and predicting treatment response. Additional important advantages include increased objectivity in interpretation, longitudinal tracking, and ability to incorporate computational models to create automated diagnostic and clinical decision support systems. Advances in computational technology, including deep learning and radiomics, open new doors for developing an imaging phenotype that may provide in-depth personalized disease characterization and enhance opportunities in precision medicine. In this review, we summarize current quantitative and radiomic imaging biomarkers described in the literature for age-related macular degeneration and diabetic eye disease using imaging modalities such as OCT, FA, and OCT angiography (OCTA). Various approaches used to identify and extract these biomarkers that utilize artificial intelligence and deep learning are also summarized in this review. These quantifiable biomarkers and automated approaches have unleashed new frontiers of personalized medicine where treatments are tailored, based on patient-specific longitudinally trackable biomarkers, and response monitoring can be achieved with a high degree of accuracy. Ophthalmology and the field of retinal diseases relies heavily on information derived from ophthalmic imaging for diagnosis, treatment and disease activity monitoring. The development of different imaging modalities, including optical coherence tomography (OCT) and ultra-widefield fluorescein angiography (UWFA), have provided incredible visualization of retinal microstructures and abnormalities, which has helped to build new insights for the management of retinal diseases, including diabetic eye disease  and age-related macular degeneration ,2.Optical coherence tomography (OCT) is a rapid, non-invasive diagnostic test that provides outstanding visualization of cross-sectional and 3D morphological characteristics in addition to high-definition anatomy. OCT has become the backbone for the diagnosis and management of retinal diseases, with more than 30 million OCT scans being performed annually ,5. Due tThere are some important limitations to consider related to current OCT technology. With a limited field of view in most widely available OCT devices, the primary imaging location is the macula, and peripheral changes may be missed, especially in early disease ,10. FurtUWFA is an emerging imaging technique which enables visualization of panretinal vascular abnormalities including leakage, microaneurysms, and nonperfusion . UWFA isOptical coherence tomography angiography (OCTA) is a major leap forward in this regard as it is completely non-invasive and provides high-resolution 3D binarized vessel maps that are objective and easy to interpret. The depth-encoded nature of the OCTA vascular data provides a unique advantage for evaluating the location of vascular abnormalities. However, current technology is primarily limited to macular pathology and can be subject to significant quality challenges, such as motion artifacts ,12,14. ACurrent imaging systems provide outstanding details of disease burdens and the impact of different retinal diseases. Traditionally, this information has been utilized in a qualitative manner and relies on an ophthalmologist’s interpretation and expertise. This inherently introduces bias and subjectivity in the assessment of these images, and therefore may limit consistency and the opportunities for precision medicine. Additionally, all of these images encode incredible amounts of data related to the underlying imaging phenotype of a given disease. These features, such as the location and type of leakage in UWFA or the reflectivity features of cysts on OCT, may carry critical information regarding the underlying pathophysiology and driving cellular pathways of a given disease ,17,18,19Recently, machine learning (ML) based algorithms has gained traction for use in several medical image processing operations such as organ segmentation , cancer Radiomics is an emerging field of medical image processing that refers to the computerized data extraction from medical images and aims to capture the subvisual image attributes that may not be identified by the human experts. It provides opportunity to physicians to interpret images better regarding individualized therapy, surveillance, diagnosis, and prognosis . These aThe boom in this image analysis space over the past decade has made it possible to automate the quantification and interpretation of ophthalmic imaging biomarkers. These computational imaging elements can then be evaluated for their role as biomarkers for disease diagnosis, prognosis, treatment initiation, and therapeutic response. For this review, we describe these measured features that are found to have clinical applications for the management of disease as “quantitative imaging biomarkers”, which may serve as objective tools for the future in the context of diabetic eye disease and age-related macular degeneration .Review Methodology. A literature search was performed using the key words “quantitative imaging”, “diabetic retinopathy”, “age related macular degeneration”, “OCT”, “OCTA”, “fluorescein angiography”, and “quantitative biomarkers” on databases, including PubMed Central and Google Scholar. Studies reporting quantitative imaging biomarkers using OCT, OCTA, and FA in diabetic eye disease and age-related macular degeneration. Studies that included only qualitative findings or that focused on pathologies other than diabetic eye disease and AMD were not included in this study.In diabetic retinopathy, increased central subfield retinal thickness (CST) and de-creased retinal nerve fiber layer thickness have been associated with increased severity of retinopathy (DR) ,30,31. FTh extraction of quantitative fluid features and the assessment of retinal multi-layer segmentation has provided insights into disease prognosis and overall longitudinal disease dynamics. A recent study confirmed quantitative improvement in ellipsoid zone integrity subsequent to anti-VEGF therapy for DME . This meUtilizing these techniques, various imaging biomarkers may be able to be linked to the underlying pathways involved in disease pathogenesis. In a recent study assessing quantitative imaging biomarkers and cytokine expression, the levels of multiple cytokines , monocyte chemotactic protein-1 (MCP-1), and interleukin-6 (IL-6)) were linked with various imaging biomarkers, such as fluid parameters and outer retinal integrity . The ideUtilizing an attention-based convolutional neural network (CNN) model using pre-treatment OCT scans that preserved and highlighted the global structures in OCT images and enhanced local features from fluid/exudate-affected regions, Rasti et al. utilized retinal thickness information for the prediction of the response to intravitreal anti-VEGF treatment . An addiBeyond evaluating for treatment need, additional studies have assessed specific retinal compartment radiomics features that may predict therapeutic response. In a recent study , the relUltra-widefield fluorescein angiography (UWFA) can capture 200° field of view (FOV) compared to conventional imaging with 30–60° FOV, enabling a more comprehensive disease evaluation. ,47 VisuaVarious biomarkers are investigated for severity grading, progression, and treatment response. Nonperfusion area, ischemic index, leakage, and microaneurysm counts have been shown to correlate strongly with the clinical severity of DR and treatment response . Ehlers The spatial distribution of DR lesions on ultra-widefield photography including MA, cotton wool spots, intraretinal microvascular abnormalities, neovascularization, and fibrovascular proliferation was investigated in a large study with 1406 eyes demonstrating a predominantly central distribution in 63% of eyes . Silva ep = 0.009) and 10% in monthly aflibercept (p = 0.18) treatment [In addition to the assessment of disease burden, quantitative feature characterization can also be used to assess treatment response. In an automated UWFA approach, intravitreal anti-VEGF therapy demonstrated significant and stark improvements in leakage index and total microaneurysm counts in DR ,58. Wykoreatment . In a prreatment . In a rareatment .Another recent study assessed the correlation between UWFA imaging phenotype and cytokine expression in eyes with DME from the IMAGINE study . The autIn addition to clinically apparent biomarkers, Fan et al. demonstrated the branching complexity of peripheral vessels and the distribution of nonperfusion areas correlated with DR severity . FractalQuantitative UWFA has also been explored in DME pathogenesis. The leakage index and MA count in the posterior pole have been associated with the presence and severity of DME . The nonQuantitative UWFA biomarkers have been explored as assessment tools for therapeutic response in eyes with DME treated with aflibercept in the PERMEATE study . AfliberBeyond characterizing the longitudinal quantitative UWFA feature alterations in response to therapy, radiomics features have been utilized to predict treatment response and durability. Prasanna et al. developed novel radiomic CIBs that characterized different morphological properties of leakage nodes and vascular tortuosity on UWFA, which were linked to the durability of anti-VEGF treatment . The disOCTA uses non-invasive OCT technology to obtain vascular structural information by assessing decorrelation in the OCT signal due to vascular flow. As a result of the depth resolution of OCTA, different chorioretinal vascular plexuses, such as the nerve fiber layer plexus (NFLP), ganglion cell layer plexus (GCLP), intermediate capillary plexus (ICP) and deep capillary plexus (DCP), have been studied using this technology. NFLP and GCLP form the superficial vascular complex while ICP and DCP form the deep vascular complex .An increased foveal avascular zone (FAZ) size is noted in patients with DR compared to normal ,70,71. RVessel density, as calculated from OCTA, has been shown to be inversely correlated with DR grade in multiple trials ,76,77. IVessel diameter index (VDI) is a representation of vessel diameter obtained by calculating a ratio of the total area of the scan occupied by blood vessels and the total skeletonized length of blood vessels in the scan. In a recent study, the VDI obtained using OCTA has been shown to positively correlate with the severity of DR and blood glucose levels ,80,81.Similar to UWFA, retinal vessel tortuosity in OCTA is another important metric that holds high potential for the evaluation of DR. Vascular tortuosity on OCTA positively correlates with the severity of DR in superficial and deep retinal vascular plexuses in moderate to severe DR . In a reNon-neovascular  age-related macular degeneration has been extensively evaluated for numerous imaging biomarkers such as intraretinal hyper-reflective foci (HRF), complex drusenoid lesions , subretinal drusenoid deposits (SDDs), and drusen burden. SD-OCT has been used to qualitatively describe these biomarkers and has confirmed that each of these features confers a greater risk of disease progress ,85. In aAutomated drusen volume quantification has been enabled by multi-layer segmentation platforms that provide isolation of the sub-RPE compartment. One study demonstrated that an increase in the drusen volume was associated with a significant increase in the risk of developing geographic atrophy or conversion to neovascular AMD . ML-enhaDL-based analysis systems have been explored to detect the presence of disease. Multiple other studies have shown the effectiveness of DL models in classifying normal versus AMD eyes from OCT images ,93. AutoIn neovascular AMD, CNV is a major cause of vision loss due to photoreceptor damage that results from exudation processes ,97. AlthIn one study aimed at characterizing CNV using quantitative biomarkers on OCTA, the CNV area and flow index using outer retinal choriocapillaris OCTA slabs for assessment of CNV characterization . The stuIn non-neovascular AMD, OCTA has been explored to study many aspects of the disease process such as drusen, reticular pseudodrusen, and geographic atrophy, in addition to exploring its utility for the monitoring of disease progression . ChoriocQuantitative imaging biomarkers derived from multiple imaging modalities may provide a critical platform for the future in providing objective and trackable metrics that enable precision medicine in ophthalmic care through the comprehensive characterization of the “imaging phenotype”, OCT imaging biomarkers provide valuable structural information of retinal layers, such as retinal compartment thickness, layer integrity maps, fluid volume, and the fluid index. UWFA and OCTA imaging biomarkers provide key information regarding the retinal and choroidal vasculature, such as measures of vessel density, ischemic area, flow voids, leakage area, leakage index, ischemic index, and the CNV area. Radiomics is an emerging field in ophthalmology and is having an increasingly high impact on personalized medicine. As the field matures in the future, a combination of different novel DL networks and advanced radiomic methods may be of high value for a more complete decision support system . The imp


The worldwide rise in prevalence of chronic kidney disease (CKD) demands innovative bio-medical solutions for millions of kidney patients. Kidney regenerative medicine aims to replenish tissue which is lost due to a common pathological pathway of fibrosis/inflammation and rejuvenate remaining tissue to maintain sufficient kidney function. To this end, cellular therapy strategies devised so far utilize kidney tissue-forming cells (KTFCs) from various cell sources, fetal, adult, and pluripotent stem-cells (PSCs). However, to increase engraftment and potency of the transplanted cells in a harsh hypoxic diseased environment, it is of importance to co-transplant KTFCs with vessel forming cells (VFCs). VFCs, consisting of endothelial cells (ECs) and mesenchymal stem-cells (MSCs), synergize to generate stable blood vessels, facilitating the vascularization of self-organizing KTFCs into renovascular units. In this paper, we review the different sources of KTFCs and VFCs which can be mixed, and report recent advances made in the field of kidney regeneration with emphasis on generation of vascularized kidney tissue by cell transplantation. The mammalian kidney is an essential organ, carrying out numerous functions such as blood filtration, regulation of water and solute balance and hormone production.Chronic kidney disease (CKD) is a progressive decline in kidney function, which can deteriorate to the state of end stage kidney disease (ESKD). Deterioration of kidney function measured by glomerular filtration rate (GFR) is accompanied by tubular interstitial fibrosis, a typical manifestation of CKD, loss of nephron cells and reduction of nephron number. The only options available for ESKD patients are dialysis and/or lifesaving kidney transplant . DialysiThe field or regenerative medicine utilizes cells, small molecules, and extra-cellular matrices to reconstitute the native tissues function . The chaSince progression of CKD is characterized by loss of kidney cells and nephrons, it would be beneficial to find a cell source which would replenish lost cells and halt disease progression, or perhaps reverse it. Multiple cell sources have been suggested for kidney regeneration in the past: fetal kidney, adult kidney, and urine derived kidney cells. Furthermore, in the past decade, protocols for generating pluripotent stem cell-derived kidney organoids (PSC-KOs) have been devised which allow the creation of autologous renal tissue . HoweverThe aim of this review is to concisely summarize the various cell sources for KTFCs and VFCs used for regenerating nephron parts and vascular components of the kidney, report on the most recent advances in cell-based methods of vascularized kidney regeneration and describe the remaining challenges in the field.Various studies in the past two decades have uncovered the complex and unique process of nephrogenesis, which culminates in the formation of a functional kidney ,10.The kidney tubules, representing the epithelial compartment, develops via reciprocal interaction of the metanephric mesenchyme (MM) and the ureteric bud (UB) . The invThe renal interstitium compartment, shown to originate from FOXD1+ progenitors, residing within the early MM plays a vital role in the differentiation of nephron progenitors . Even thThe vasculature is the third critical compartment of the kidney in both health and disease. The renal vasculature can be divided into major blood vessels , glomerular vasculature and the peritubular capillaries (vasa recta). The origin of these compartments in embryonic kidney development is still under debate, and two main theories exist as to its origin. The first theory argues that vascularization is an extra-renal process driven by angiogenesis–pre-existing vessels sprout new vessels which penetrate the MM and vascularize the vascular cleft of the proximal region of nascent nephrons during the s-shape phase. The second theory argues that vascularization is an intra-renal process driven by vasculo-genesis, in which resident epithelial stem cells  differentiate and form blood vessels around the developing nephrons. This process is thought to be driven by the secretion of VEGFA by the podocyte progenitors in the vascular cleft in the proximal s-shaped body . Recent Theoretically, the fetal kidney is the ideal source for regeneration, since by definition, it harbors all progenitor populations needed to generate a kidney. Indeed, human FK precursors have been transplanted to form miniature kidneys that generate dilute urine . HoweverAutologous adult renal tissue is an attractive cell source since it does not have the same ethical limitations as the FK and, more importantly, potentially allows for autologous cell therapy. The adult kidney harbors heterogenous lineage restricted precursor cells (LRPCs) which can clonally expand to replace damaged cells after injury . These LIn the past decade, multiple protocols for generating kidney organoids from pluripotent stem cells (PSCs) have been developed ,43,44,45As in the FK, protocols for isolation and expansion of NPCs from PSC-KOs have been devised . Single-When cells/tissues are transplanted into a host they must be provided with oxygenation, nutrient delivery, and waste product removal. This can be accomplished via angiogenic processes in which the host’s vasculature connects or surrounds the transplanted cells/tissues. However, evidence shows that without support of encircling smooth muscles  these blood vessels are short lived . This meIt is also important to consider the resident vascular and stromal cells which are present in primary fetal and adult tissues, which might also be sorted out, propagated, and utilized. The kidney vasculature has a distinct transcriptional profile ,65 whichFor regenerative capacity testing, KTFCs and VFCs are often mixed in ECM hydrogels such as Matrigel and injected, usually into the kidney . There aWhen mixing fetal KTFCs with VFCs to form vascularized kidney tissue, an intuitive strategy is to use resident VFCs from the native FK Mice fetal kidney organoids consisting of isolated endothelial precursors (CD31+ and/or Flk1+) mixed with isolated MM cells  to form kidney organoids which upon transplantation vascularize, mainly by the donor . InteresOur group has recently shown that co-administration of human AK cells, with ECFCs and MSCs, increases the tubulogenic capacity of injected kidney cells, upregulates self-renewal and mesenchymal to epithelial transition markers and facilitates connection to the host’s vasculature . AlthougTaniguchi’s group reported an ex-vivo method for generating vascularized organ buds by combining pluripotent stem cell-derived tissue progenitors , as well as tissue fragments, with ECs and MSCs. Upon intracranial transplantation in mice, the organ buds successfully integrated into the host . NotablySharmin et al. transplanted iPSC-derived kidney organoids with HUVECS and MSCs to the mouse kidney capsule . After 2The first approaches to kidney regenerative medicine utilized the embryonic kidney stem cell compartment in the FK, the MM, which gives rise to vascularized nephrons. Vascularization is a key component which will have to be addressed in order to allow regenerated kidney parenchyma to produce and secrete urine. Blood vessels need to invade the glomerulus to establish blood filtration and surround the other segments of the nephron to allow additional fine tuning of solute transport. It is important to note that the various KTFCs reviewed in this paper have different regenerative capacities: KTFCs from FK and PSC-derived kidney organoids can generate the entire nephron, while KTFCs from AK are lineage restricted and produce segment specific tubules. This constraint must be taken into consideration when planning vascularization and co-engraftment with VFCs and supporting cells. KTFCs from AK will not be able to reconstitute glomerular filtration since glomeruli are usually lost during culture. Nevertheless, the vascularized tubular structures created might add a layer of functionality to CKD patients via paracrine effects and absorption and secretion of solutes.KTFCs from “immature” sources such as the fetal kidney and PSC-KOs promote host vascularization via VEGF secretion and endogenous VFCs. Most importantly, engrafted organoids exhibit blood vessel invasion to the glomeruli, a landmark of functional renal tissue. The AK cell source is purely epithelial and thus benefits from co-administration with VFCs, showing increased tubulogenic capacity. In case of transplantation of KTFCs from any source into CKD kidneys it is reasonable to consider that vascular dysfunction occurring in CKD would dampen host vascularization and hence mixing cells to achieve donor vascularization is likely to be required. The ultimate strategy for generating renovascular units, with glomerular blood filtration and vasa recta, is to engraft KTFCs with VFCs, which could be derived by differentiating PSCs into a vascularized kidney organoids in-vitro or in-vivo. To this end, further priming for vascularization could be addressed by providing mechanical cues and growth factor gradients promoting VFC proliferation and differentiation. This could lead to the development of a universal off the shelf kidney regenerative cell source for mixing KTFCs and VFCs, especially with the ability to modulate immunogenicity via CRISPR-CAS9 gene editing ,78.