Human brain aging is characterized by the gradual deterioration of its function and structure, affected by the interplay of a multitude of causal factors. The sleep, a periodically repeating state of reversible unconsciousness characterized by distinct electrical brain activity, is crucial for maintaining brain homeostasis. Indeed, insufficient sleep was associated with accelerated brain atrophy and impaired brain functional connectivity. Concurrently, alteration of sleep-related transient electrical events in senescence was correlated with structural and functional deterioration of brain regions responsible for their generation, implying the interconnectedness of sleep and brain structure. This review discusses currently available data on the link between human brain aging and sleep derived from various neuroimaging and neurophysiological methods. We advocate the notion of a mutual relationship between the sleep structure and age-related alterations of functional and structural brain integrity, pointing out the position of high-quality sleep as a potent preventive factor of early brain aging and neurodegeneration. However, further studies are needed to reveal the causality of the relationship between sleep and brain aging. Aging is accompanied by a gradual loss of function and structural degeneration in all organ systems . In the Therefore, understanding the processes underlying brain aging is crucial to preventing the development of age-related diseases, and potentially also neurodegenerative disorders, opening a window for eventually targeted prevention and early treatment—the holy grail of a vast segment of neuroscientific research . ParticuThe sleep of insufficient quality and length does not only immediately reduce the elimination of toxic waste products  and nextFilling the void in the currently available literature, this review focuses on the link between brain aging and sleep—one of the key modifiable risk factors—through the viewing glass of various neuroimaging and neurophysiological methods.There is wide agreement among neuroimaging studies that cortical and subcortical GM volume decreases with age , with suIn addition to trophic changes, brain functional connectivity changes with age. In conformity with morphological tissue loss, the effects of aging were most consistent in highly overlapping regions of DMN and medial temporal lobe subsystem . The funThe sleep of insufficient length and quality is associated with accelerated brain atrophy . ConcernThe integrity of WM tracts is also affected by sleep . Even thFurthermore, the functional connectivity of neuronal networks changes during the sleep-wake cycle . Sleep dAs previously mentioned, sleep has an ample impact on the structural and functional changes of the brain during the life course—both sleep duration and quality are crucial .Two types of sleep are distinguished –rapid-eye movement (REM) and non-rapid-eye movement sleep (NREM) sleep. NREM is further divided into three stages , based on distinct electroencephalographic features . These sSimilar to brain morphology and function, sleep structure is also substantially impaired in senescence. Total sleep time, and proportionally mainly N3 sleep, becomes progressively shorter and more fragmented with subsequent diminution of sleep efficiency and quality . At the The neuronal activity of the thalamocortical loop plays a key role in SS generation during sleep . CorrespAdditionally, the integrity of WM and GM seems to moderate the effect of sleep on memory consolidation. The integrity of temporal WM tracts and hippocampal GM volume and its neural activity were predictive of SS impact on overnight motor memory consolidation . This moThe current review presents and attempts to synthetize the following conclusions:(1)Brain atrophy and disruption of functional neuronal connectivity are hallmarks of brain aging. This conclusion is supported by a number of neuroimaging studies . However(2)Aging is associated with sleep shortening, fragmentation, and deterioration of its quality. Both, NREM and REM sleep are affected . Even th(3)Individuals with inadequate sleep length and/or quality are more commonly affected by brain atrophy and have lower cerebral functional connectivity. Short sleep duration and poor sleep quality are longitudinally associated with an increased cortical thinning rate . During (4)Simultaneously lower GM and WM integrity is predictive of poorer sleep. Studies using mediation analysis models suggest that neuronal sleep oscillations, a proxy of the quality of sleep, are influenced by cortical integrity . SpecifiTaken together, sleep and brain structure and function are strongly interconnected. Brain structure impacts the interindividual variability of sleep oscillations, while sleep significantly affects brain metabolism, gene expression and, consequently, brain tissue integrity. Ergo, possible improvements in the duration and quality of sleep may represent a potent modifiable factor in the prevention of aging-related brain changes, ultimately also dementia and other neurodegenerative diseases. Considering the ubiquitous chronic sleep deprivation, we are in dire need of longitudinal multimodal studies, which could shed further light on the causality of the relationship between age-related alterations and sleep disturbances and possibly also on the extent and further qualities of the proposed bidirectionality.In conclusion, the currently available literature implies a plausible and non-negligible interconnection between age-related alterations to the central nervous system integrity and sleep structure. Nevertheless, the true underlying causality of the relationship between sleep and aging remains elusive. Does poor sleep quality or quantity lead to accelerated brain atrophy, microstructural, and functional changes generally seen in aging? Or is the direction of the association inverse—is poor sleep quality only a secondary symptom of aging-related cerebral changes? Can we slow down or prevent some of the brain changes associated with aging by improving sleep quality and duration? Unfortunately, we do not know. Thankfully, the field of sleep research is going through a rapid evolution in available study methods, with much to be expected from novel polysomnographic approaches and imaging-derived metrics.VK reviewed the literature and wrote the manuscript. DK, MB, and PF read and revised the manuscript. All authors approved the final manuscript.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.


Despite extensive research effort in recent decades, the underlying mechanisms involved in the reprogramming of stromal cells in chronic inflammatory conditions and cancer are still ill-defined. This research topic includes original research and reviews of important concepts on inflammatory microenvironment remodeling and 3D models.Adipose progenitor cells constitute a small and heterogeneous group of cells that can be modulated by mature adipocytes as well as by resident immune cells to determine their function and their differentiation potential. Several pathologies can destabilize the relationships created and break the equilibrium. In their review, Pyrina and others summarize the current knowledge about various factors and different immune and stromal cell populations that fate decisions of the adipose progenitor cells in the context of obesity-related inflammation. They define how in the course of obesity the pro-inflammatory microenvironment through multiple contact- and paracrine-mediated interactions shape the differentiation of preadipocytes, towards a pro-fibrotic transcriptional mode that in turn promotes fibrosis.The NR4A receptors are potent sensors of changes in cellular microenvironment allowing the control of physiological and pathological processes. In their review, Murphy and Crean illustrate how this subfamily of nuclear receptors can be considered as transcriptional regulators in mesenchymal stromal cells, cell cycle progression and cytokine production to control local immune response in physiological or in chronic inflammatory conditions. Authors made a focus on recent advances in the pharmacological control of these receptors.Gastrointestinal cancers are among the most frequent cancer-types worldwide and have been strongly linked with chronic inflammation. For that reason, current studies and therapies focus on cancer cells but also on the cells present in the tumor microenvironment (TME). Melissari and others review the role of cancer associated fibroblasts (CAFs), a major component of the TME, in tumor initiation, promotion and metastasis. They focus on the mechanisms that drive fibroblast reprogramming in gastrointestinal cancers, as well as on recent advances in therapies and patient prognosis.The role of tumor infiltrating neutrophils in cancer progression of gastric cancer is highly appreciated . Zhang aIn an additional publication focusing on CAFs, the emerging grasp of the TME is consistently unfolding even in cell-endogenous and evolutionally conserved mechanisms. In their research article, the group led by Ruth Scherz-Shouval have worked on the synchronized cellular circadian clock which was previously reported to facilitate a plethora of processes including gene regulation and homeostasis in the cell. The authors were able to reveal a novel aspect of the circadian clock disrupted during tumorigenesis and metastasis as cancer cells were compared to normal fibroblasts. Out of many related genes, Period 2 (Per2) was identified as a cardinal hub associated with this disruption. When Per2 was eliminated or knocked-out, the authors were able to abolish the metastatic progression. Notably, the expression of Per2 was found mainly in CAFs, a critical component of the TME.The role of fibroblasts is further described in cutaneous T-cell lymphoma (CTCL) which refer to a group of lymphoproliferative diseases characterized by the accumulation of malignant T cells in chronically inflamed skin lesions. In their review, Stoleranco and others describe the crosstalk between fibroblasts, keratinocytes and malignant T cells and how they contribute to CTCL progression. Among the complex signaling networks, authors focus on the activation of STAT proteins, the development of a Th2 inflammatory microenvironment, neovascularization of the tumor tissue and changes in the skin architecture.Notably, there is growing evidence describing the role of the TME in the development and progression of hepatocellular carcinoma (HCC) . HCC typTwo studies in this special issue highlight the advantages of 3D models to study human pathology. Vergnolle and others characterized morphological and functional phenotypes of colonic organoids from Inflammatory Bowel Disease (IBD) and control patients. The authors demonstrated that IBD organoid cultures preserved an inflammatory phenotype and that clinically used treatments reduced some parameters associated with this inflammatory phenotype. This study highlights that IBD patient organoids constitute a reliable human pre-clinical model to study IBD pathology and investigate new strategies targeting epithelial repair. In the second study, Haykal and others, depict the importance of using and developing new models and tools taking into consideration tissue complexity. A focus is made on organotypic models allowing to study cancer progression in complex settings and offering potential benefits for personalized medicine.Altogether, these topical articles highlight the complex interactions between parenchymal cells and the surrounding stroma in different contexts. Besides, the advantages of 3D models to study the human pathophysiology of chronic inflammatory diseases and cancer and for pharmacological drug testing are underlined. In the era of personalized medicine, a profound knowledge of tissue microenvironment could improve prognosis and therapy in clinical practice.


The visual system is a source of sensory information that perceives environmental stimuli and interacts with other sensory systems to generate visual and postural responses to maintain postural stability. Although the three sensory systems; the visual, vestibular, and somatosensory systems work concurrently to maintain postural control, the visual and vestibular system interaction is vital to differentiate self-motion from external motion to maintain postural stability. The visual system influences postural control playing a key role in perceiving information required for this differentiation. The visual system’s main afferent information consists of optic flow and retinal slip that lead to the generation of visual and postural responses. Visual fixations generated by the visual system interact with the afferent information and the vestibular system to maintain visual and postural stability. This review synthesizes the roles of the visual system and their interaction with the vestibular system, to maintain postural stability. Postural control requires continuous regulation of information from three systems- the visual, the vestibular, and the somatosensory . A key pSelf-motion and motion of an object in the environment whilst a person is stationary cause a similar visual stimulation . For exaThere are a number of reviews discussing the roles of the visual system and the vestibular system in postural control . HoweverThe visual system consists of the central visual system (fovea) and the peripheral visual system. The central visual system recognizes objects and object motion, whereas the peripheral vision is sensitive to moving scenes and dominates the awareness of self-motion and postural control . To mainOptic flow is the pattern of motion of the external world over the retina and forms a part of the afferent information to the visual system . For exaThe vestibular system comprises the peripheral and central vestibular systems and serves a wide variety of functions such as postural control, gaze stabilization, conscious perception, autonomic regulation, and navigation. This review will focus on its role in postural control and gaze stabilization . It mediThe peripheral vestibular receptors provide information about the motion of the head in three dimensions. The central vestibular pathways use this information to control the reflexes and perception of self-motion . The vesThe vestibulo-ocular reflex (VOR) is a gaze stabilizing reflex which stabilizes the retinal image by rotating the eyes in the opposite direction to head movements . It is dVisually perceived orientation of the environment provides cues to verticality but can sometimes confound orientation. To interpret visual cues properly, the contributions of object-in-world and eye-in-world orientations from the retinal images must be reconciled to ensure an accurate perception of verticality . The vesThe generation of vestibular reflexes in response to visual input signifies an intimate relationship between the visual and the vestibular system such as seen in the optokinetic reflex. This reflex responds to input from the otolith organs and regulates eye position during head rotation and tilting . It is aThere are three sections: (1) optic flow and postural control: this section describes how optic flow is generated, what it is used for and its role in postural control, (2) retinal slip, vestibulo-ocular reflex, and postural control: this section emphasizes how the retinal slip is interpreted and its interaction with the vestibular system to maintain postural control, (3) visual fixations and postural control: this section incorporates the role visual fixations play in postural control by interaction with the optic flow and the retinal slip. Finally, visual-vestibular interaction is discussed at the neuronal level.When a person moves in an environment, it is necessary to differentiate self-motion from external motion to maintain postural stability . This diMovement of an observer in a stationary environment is interpreted as self-motion as it generates patterns of optic flow specific to self-motion . In the The optic flow pattern created during self-motion is not consistent throughout the visual field . During In addition to optic flow, vestibular signals are important for inferring self-motion . The visRetinal slip is the afferent signal used to generate visually evoked postural reactions . These pDuring self-motion, objects within the visual scene move on the retina generating retinal slip, this can lead to a blurry perception of the scene and the object. To avoid this, visual and vestibular systems co-function to compensate for retinal slip by generating compensatory eye movements . The eyeThe complexity of retinal slip increases when the observer moves closer to an object, or the object lies at an angle to the direction of motion. To maintain the body in a stable position, retinal slip must be minimized . To miniThe functioning of the VOR depends on three significant context variables; the head movement characteristics (known as stimulus context), fixation during head movements , and the motion of visual target  . The heaFor maintained fixation during head movement, VOR compensates for both translational and rotational components. Compensation is dependent on fixation distance. Fixation on a distant target requires little eye movement, as the object gets closer a larger amplitude of ocular responses is generated .The mode of visual-vestibular interaction is dependent on whether the visual target is stationary or moving. If a visual target is stationary, the VOR efficiently compensates for any sudden perturbations of the head in space. Activities such as locomotion achieve gaze stability by activating semi-circular canal afferents through head movements, triggering the VOR. The eye movements generated are so accurate that there is no retinal slip, maintaining high visual acuity and gaze stability .Visual fixations keep our eyes fixed on a target while viewing a scene. Visual fixations occur between saccades, contribute to 80% of the visual experience and are essential for visual processing . Within Visual fixations have an important role in reducing optic flow, minimizing retinal slip, and suppressing the optokinetic response . MinimizLarge field visual motion typically generates the optokinetic response . Such stVisual fixations have a key role in maintaining postural stability as visually fixating on a target decreases postural sway . Two theThere is a large literature around neuronal control of visual-vestibular interaction. For this review, we are restricting discussion to brain areas involved in visual-vestibular interaction, neuronal mechanism for visual fixation control and interaction between these areas to maintain gaze and postural stability.Visual-vestibular interaction is necessary to estimate and continuously update the body position in space and to distinguish self-motion from external motion. Explanation of this interaction has been widely studied in Macaque monkeys. The exact neural mechanisms for visual-vestibular integration in humans is less well understood . Early sThese findings led to the current hypothesis of reciprocal visual-vestibular interaction based on reciprocal inhibition . Visual-A large number of areas have been associated with resolving perceptual conflicts . These iAdditionally, the existence of visual targets in the environment requires a combination of eye and head orientation to achieve gaze stability. The visual-vestibular interaction to shift gaze toward a target and then maintain fixation is regulated by omni-directional pause neurons (OP neurons), located in nucleus raphe interpositus of the paramedian pontine reticular formation . These nThe input to the OP neurons is a weighted sum of the vestibular and visual inputs . This coTherefore, there is a continued interaction between visual and vestibular systems for postural control to maintain body and eye stability during various transitions involving head movements and constant visual motion.The visual information regarding movements of self and objects in the environment is fundamental to postural control. Information from the optic flow patterns helps differentiate self-motion from external motion. Concurrent information of self-motion is also provided by the vestibular system using angular and linear acceleration of head in space. This information is necessary in instances when information from optic flow generates a false perception of self-motion known as vection or stimulates an optokinetic response. Optic flow patterns generate retinal slip on the retina constituting the main afferent signal to generate visually evoked postural reactions. To maintain visual and postural stability, the visual system, and the vestibular system co- function by generating TVOR and OFR’s respectively to stabilize the image on retina. Stabilization of retinal image eliminates retinal slip, providing information to the nervous system to maintain an upright stance by generating compensatory postural sway.A key determinant of visual and postural stability is visual fixations which keep eyes fixed on a target while viewing a scene. Visual fixations suppress the optic flow and minimize retinal slip by maximizing the peripheral vision and suppressing the generation of vection. They also have a major role in suppressing the optokinetic response which can destabilize an observer. Further, they maintain visual stability during tracking a moving target by suppressing the VOR.The current review outlines how visual-vestibular interactions enhance postural stability by interpreting the head’s position and generating eye movements accordingly, which helps differentiate self-motion or external motion and achieve gaze stabilization and postural control.SC drafted the manuscript. DT and NS provided Ph.D. supervision for SC. All authors critically revised the manuscript read and approved the final manuscript.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.


The syndrome of uremic toxicity comprises a complex toxic milieu in-vivo, as numerous uremic substances accumulate and harm the organ systems. Among these substances, toxic and non-toxic players differently interfere with human cells. However, results from animal experiments are not always compatible with the expected reactions in human patients and studies on one organ system are limited in capturing the complexity of the uremic situation. In this narrative review, we present aspects relevant for cellular toxicity research based on our previous establishment of a human spermatozoa-based cell model, as follows: (i) applicability to compare the effects of more than 100 uremic substances, (ii) detection of the protective effects of uremic substances by the cellular responses towards the uremic milieu, (iii) inclusion of the drug milieu for cellular function, and (iv) transferability for clinical application, e.g., hemodialysis. Our technique allows the estimation of cell viability, vitality, and physiological state, not only restricted to acute or chronic kidney toxicity but also for other conditions, such as intoxications of unknown substances. The cellular models can clarify molecular mechanisms of action of toxins related to human physiology and therapy. Identification of uremic toxins retained during acute and chronic kidney injury enables further research on the removal or degradation of such products. Human cell models must be developed alongside the disease of interest. The syndrome of uremic toxicity includes complex variables to be considered for creating in-vitro conditions adequate to the uremic state. During acute and chronic kidney failure, numerous substances are retained in higher concentrations in uremic patients and are denominated as uremic toxins ,2. It isUremic patients suffer from various and different symptoms depending on the degree of toxicity, but also on individual pathological reactions and responses to such substances. A negative effect of various toxins may be identified by various cell models or exposure of substances to animals . HoweverThe exposure of specific toxins to humans meets ethical limitations. However, such exposure was made in 1972 when urea was added to the dialysate in patients with far-advanced renal failure. There were no major toxicity findings noted in that study if the dialysate concentration was below 30 mmol/L, while the fast removal by dialysis from these levels caused side effects .Mechanistic studies, using cell models derived from one organ system, can identify and characterize single uremic toxins; however, they are naturally limited in capturing the complexity of the uremic situation. The uremic situation comprises a dynamic environment that is altered by the physiological response of the cells, their pathological decay, and the clinical situation of the patients, e.g., their medication. Our team developed a human spermatozoa-based model for the evaluation of toxic effects on cell viability and cell motility . SpermatOur human model combines the analysis of apoptotic and mitochondrial cell function. Firstly, our model includes a visual cell death evaluation resembling a putative expression of cell membrane stability in an analogy to models evaluating the median lethal dose (LD50). Secondly, our model includes a cell motility evaluation resembling putative interference of motility patterns and myofibrillar functions. The integration of cellular viability with its functional response allows for a more stringent resemblance of the uremic toxicity situation.This narrative review aims to give general guidance for the establishment of cellular toxicity models. In the following review, we present four key aspects based on our previous establishment of a spermatozoa-based cell model that may expand the impact of a cell model to study uremic toxicity .max) [The uremic milieu comprises a growing number of retained uremic substances. By the current identification of more than 100 uremic substances , the celmax) , we obsetoxins based on their accumulating state in serum. This accumulation in serum does not necessarily associate with a high degree of cellular toxicity. We reason that experiments should specifically aim to uncover the protective roles of uremic substances. Ideally, a developed cell model can identify a toxic effect but also a putative protective effect of a substance. Holistic functional cellular readouts are considerably suitable to characterize cell states beyond two categories, e.g., dead versus alive. The cellular readout analysis comprises a quantitative or “gray” scale and can give more detailed guidance in assessing the effect of a substance in the broad spectrum between toxic and supposedly protective. Cellular motility serves this aim since both states of substance-induced hypomotility versus hypermotility represent a functional continuum that indicates the effect of a substance. Following this approach, we could identify uremic retention solutes from all three of the molecular classes that are assigned to the toxic category (as defined by hypomotility) and those that are assigned to the protective category (as defined by hypermotility)  .max (the highest concentration observed in a uremic patient) in an analogous manner for drugs. This enables us to investigate the real serum toxicity of patients in pre-dialysis conditions. Furthermore, we suggest including drugs within the identical experimental design of testing uremic substances. In order to estimate the degree of toxicity of a serum-accumulating drug, it is critical to compare it directly to the effect of a uremic toxin.Drugs can interfere with human physiological function beyond their original targets. This is especially important in the uremic situation as the serum levels of drugs can increase significantly in more progressive pre-dialysis conditions. We reason to incorporate medication conditions into uremic cell models in addition to a significant number of uremic toxins (see 2). In detail, we recommend transferring the methodology of applying uremic substances in CThe described model also allows for the separate investigation of drug toxicity, such as applicability to screen for chemotherapy induced toxic effects. These investigations can also be performed on the biological fluid from patients exposed to cytotoxic drugs prescribed by various indications.max as a positive control among multiple testing conditions. The positive control condition resulted in rapid cell death and the cease of physiological motility. Such observed effects can underline the sensitivity of a toxic assay and its applicability to the desired clinical aim of hemodialysis.The implementation of a clinical aim into an investigational analysis is central to propel the translation of experimental results into diagnostic and therapeutic application. Uremic toxin research can profoundly contribute to the application and development of hemodialysis procedures when experimental conditions are linked to the real-world clinical state. One applicable step is to apply positive controls within uremic toxin experiments that are based on the uremic state of chronic kidney patients before hemodialysis. In our study, we applied a mixture of uremic toxins in Cnon-response to hemodialysis? Studies on these patients may even help to identify novel uremic toxins.Hemodialysis can be directly integrated into the experimental set-up, i.e., a comparison between the state of patients before and after treatment. This enables the ultimate identification of the effects caused by hemodialysis (or its detoxification) and moreover, can reveal whether the hemodialytic procedures may fail to do so in individual patient cases. In our study, we directly compared the serum pre- and post-hemodialysis of chronic kidney patients. Notably, we identified one patient that did not exhibit a loss in serum toxicity after the hemodialytic procedure. Ultimately, experiments that identify non-profiting or outlier patients are valuable. These patients can be of outmost significance to follow-up with mechanistical studies. Does a physiological or clinical attribute of this patient contribute to the observed urea is used for monitoring the effect of hemodialysis, which does not show good predictive value for toxicity monitoring [Experimental uremic studies should aim to develop new hemodialysis parameters. Currently, the calculation of Kt/Vnitoring . We hypoThis commentary article presents aspects based on a human spermatozoa model that can help to create further cell models relevant for the investigation of clinical toxicity, such as in uremia. Overall, we favor to study the uremic milieu with the following parameters: (i) inclusion of a wide number of uremic substances, (ii) selection of a cellular readout that extends toxic versus non-toxic categories, (iii) consideration of the treatment of the patients, i.e., drugs, and (iv) early translation in order to improve hemodialysis. The integration of these steps may result in the creation of a more in-vivo-like uremic environment of a cell model.Multiple studies report on cell death due to uremic toxicity as a driver for physiological decrease after exposure to toxins ,28,29. IFewer models investigate the uremic effects on holistic cellular functions that maintain the physiology of organisms. For example, cellular motility is crucial for various forms of cellular communication, such as immune responses  or humanIn our clinical pilot study, we show that hemodialysis reduces the mitochondrial toxicity in the serum of the patients . This prWe envision that cellular models of uremic toxicity will level-up with the concrete clinical situation of chronic kidney disease patients. The key aspects for the establishment that are presented here may enable us to build cell models suitable for uremic toxicity research.


The human fascination with wild and exotic animals has existed for millennia . The firIn this Research Topic of five peer reviewed articles, two relate to clinical practice, two are skull-based studies, and the final study describes a new experimental model utilizing degus.Since teeth and jaw bones have been preserved in museums for decades, this source of information is invaluable to the veterinary dentist who deals with wild and exotic species. Several well-constructed studies have been published on a variety of animals before –28. In fRickert et al. report on the temporomandibular joint (TMJ) pathology and the unique TMJ anatomy of wild carnivores in the western United States. They evaluated 5,011 animals belonging to 13 species and found TMJ osteoarthritis (TMJ-OA) to be the most common pathology present, possibly linked to masticatory forces. The authors call for ecological studies to describe the function of the TMJ during feeding and fighting in order to correlate function with the pathology seen.Landy et al. utilized 28 skulls to describe the unique skull and dental pathology of an iconic Australia species, the Tasmanian devil (Sarcophilus harrisii). This study demonstrates the frustrations of retrospective studies utilizing skulls where metadata  are often absent and detracts from the ultimate value of such studies. The fact that a number of dental anomalies and pathologies were described on these wild animals is significant. It is the belief of the lead author that comparative studies between captive and wild specimens provide a deeper understanding of the pathological processes present in the captive environment and the opportunity to correct the poor husbandry practices that perpetuate such problems.The second article by Clinical articles in the field of veterinary dentistry of wild and exotic animals are often limited to single animal case reports, while case series with good follow-up evaluations are far less common –34. The Primožič et al. indicates the need for and describes the outcome of closed extractions of fractured canine teeth in 18 domestic ferrets (Mustela putorius furo). Although 50% of the fractured teeth had radiographic evidence of endodontal disease, poor correlation between pulpal health  and radiographic evidence of endodontic disease was noted. The closed extraction, with extraction sites left to heal by second intention resulted in uncomplicated and rapid healing. At follow-up eight ferrets (44.4%) developed maxillary lip entrapment by the mandibular canine tooth, while two (11.1%) had mild lip trauma that did not require any further treatment.The first clinical articles by Steenkamp et al. documented the presence and progression of a unique palatal pathology in 256 cheetahs (Acinonyx jubatus). Apart from this large cohort, a further five individuals were examined every 6 months from 7 months-of-age till 25 months-of-age. Cheetahs have deep indentation of the palatal mucosa where the mandibular premolar and molar teeth make contact with it. At the medial aspect of the maxillary fourth premolar tooth, there is a large indentation also in the palatine bone. These anatomical structures have previously been described  in an experimental study. The authors assessed the effect of chronic high dietary phosphorous levels on the mandibular ventral border, the volume of this bone apical to the fourth premolar tooth as well as the first and second mandibular molar teeth utilizing micro- computed tomography (micro-CT). Seventeen months after the start of the experiment, the degus on the high phosphorous diet had poor body condition scores, poor enamel covering the incisor teeth as well as irregular ventral mandibles with bony swellings. They also had a higher incidence of dental disease with elongation of the roots of the fourth premolar and first and second molar teeth. In addition, they had significantly thinner bone overlaying the apices of these teeth.The final article in our Research Topic by This Research Topic outlines the dilemma we have in wild and exotic dentistry today. We can produce good skull-based studies and case series, but evidence-based work is still scarce. Studies with larger cohorts should be encouraged through better collaboration of veterinary dentists globally. When dealing with wildlife, follow-up evaluations may indeed be difficult or impossible. The few cases where such good follow-up evaluations are available should be published to guide future work in this field.GS drafted the editorial. AT and AN contributed to the editorial. GS, AT, and AN amended the editorial. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Precise delivery of therapeutics to the target structures is essential for treatment efficiency and safety. Drug administration via conventional routes requires overcoming multiple transport barriers to achieve and maintain the local drug concentration and commonly results in unwanted off-target effects. Patients’ compliance with the treatment schedule remains another challenge. Implantable drug delivery systems (IDDSs) provide a way to solve these problems. IDDSs are bioengineering devices surgically placed inside the patient’s tissues to avoid first-pass metabolism and reduce the systemic toxicity of the drug by eluting the therapeutic payload in the vicinity of the target tissues. IDDSs present an impressive example of successful translation of the research and engineering findings to the patient’s bedside. It is envisaged that the IDDS technologies will grow exponentially in the coming years. However, to pave the way for this progress, it is essential to learn lessons from the past and present of IDDSs clinical applications. The efficiency and safety of the drug-eluting implants depend on the interactions between the device and the hosting tissues. In this review, we address this need and analyze the clinical landscape of the FDA-approved IDDSs applications in the context of the foreign body reaction, a key aspect of implant–tissue integration. Controlled and target-specific drug delivery is of critical importance in modern medicine. Therapeutic windows of conventional oral and intravenous drug administration routes are often limited by the undesirable side effects on non-target tissues and suboptimal first-pass metabolism . MoreoveThe implantable drug delivery system (IDDS) is a medical device that can be surgically placed inside patient tissues to introduce a therapeutic substance and improve its efficacy and safety by controlling the rate, time, and place of drug release in the body . IDDS reThe following features are most commonly listed as the advantages of IDDS: (1) precise distribution of the drug to the target tissue without bioavailability and first-pass metabolism concerns that allows reduction in the active dosage; (2) minimization of side effects due to lower active substance systemic concentrations and absence of risk of incorrect drug administration; (3) prolonged and dose-controlled delivery of the drug, which makes the therapy independent of patient compliance ,8,9. Mor125 delivered to the myocardium by the “Silastic” IDDS. While the desired antiarrhythmic effects have not been achieved, this work revealed both a great potential of IDDSs for the targeted administration of therapeutics as well as a serious challenge related to the drug distribution from the implant to the tissues and the role of fibrotic reaction at the implantation site.It is considered that the first applications of IDDSs were subcutaneous implantations of hormone-containing pellets in cattle and poultry in the 1930s ,12. Bish®, based on Folkman’s and Long’s “Silastic” capsule schematics, obtained Food and Drug Administration (FDA) approval ..SynchroMmorphine . Saline-morphine . As a remorphine ,67.Intera 3000 (or Codman 3000) presented a novel design model, an infusion pump that did not require a battery as it operated via a gas–liquid propellent system driven by body temperature  and the subsequent approval of aflibercept  for treatment of wet age-related macular degeneration (AMD) . IntraviIntraocular implants, which are usually classified as non-biodegradable and biodegradable IDDS devices, are transplanted directly into the vitreous body. The installation of the IDDS device is invasive and can cause complications similar to those caused by intravitreal injections .Typically, non-biodegradable IDDS devices entrap a drug within a reservoir surrounded by non-biodegradable release membranes . They arVitrasert was approved by the FDA for the treatment of cytomegalovirus (CMV) retinitis [Vitrasert implant was withdrawn from the European market for human use in April 2002 by the European Medicines Evaluation Agency due to ocular complications  [In 1996, the non-biodegradable implant etinitis . The impetinitis . The impetinitis . The Vitachment) .Retisert implant is used for the treatment of chronic non-infectious posterior uveitis. The intravitreal implant received FDA approval and became widely commercially available in 2005. Retisert is composed of a central core consisting of fluocinolone acetonide (FA) compressed into a 1.5 mm diameter pellet coated with a nonpermeable silicone capsule featuring an orifice to allow drug release. This device is implanted into the vitreous humor. It is also attached to a PVA suture tab and coated with extra PVA and silicon layers with a drug diffusion port, which delivers the medicament over ≤2.5 years but must then be removed [Retisert implants tend to dissociate, leading to intraoperative complications, including posterior retinal tear and limited suprachoroidal hemorrhage [The  removed . Retisermorrhage . The mosmorrhage . Based omorrhage . Additiomorrhage ,86.Iluvien implant  but provided the same release profile for 3 years [Later, a very similar IDDS,  3 years .Ozurdex dexamethasone drug delivery system  are ocular hypertension and cataracts [Ozurdex implant in the vitreous cavity, and the migration of implant into the anterior chamber [The y system d is a bi6 months . Sustainataracts . Clinicaataracts ,98. Epir chamber ,101,102.Dextenza  is approved for the treatment of pain and inflammation following cataract surgery, during which 0.4 mg of sustained release dexamethasone insert is placed inside the tubule to ensure consistent and uniform drug delivery to the ocular surface for 30 days after a single dose [gle dose . The dexgle dose . The mosSusvimo, a surgically fixed permanent refillable implant, for the continuous release of the ranibizumab, a monoclonal antibody against vascular endothelial growth factor (anti-VEGF) to treat neovascular AMD [BIM ring, a Brimonidine intravitreal biodegradable implant for the treatment of dry AMD and retinitis pigmentosa with six months release [Recently, the FDA approved several new ocular IDDSs. These include ular AMD . The devular AMD . Interes release . Due to I-vation, a non-biodegradable polymeric implant, was intended to release triamcinolone for at least two years in patients with diabetic macular edema. This is helix-shaped, non-biodegradable implant was coated with triamcinolone. It was designed to release the drug over a period of 36 months in patients with diabetic macular edema. However, the device caused complications including increased intraocular pressure and cataracts and was terminated after phase two of clinical trials [Advances in biomaterials and nanotechnology have led to major growth in the research of ocular implants. For example, l trials .Verisome technology, a fully biodegradable liquid form of an active substance in the aqueous sphere. Verisome-based, dexamethasone-containing DEXYCU obtained FDA approval to treat postoperative inflammation in 2018 [Verisome (IBI-20089) can be applied to deliver various types of drugs, including proteins, up to one year [A different approach for prolonged drug delivery was realized with  in 2018 . However in 2018 . Verisomone year .Renexus, is undergoing clinical trials for the treatment of macular telangiectasia and glaucoma. Genetically modified cells deliver human ciliary neurotrophic factor (CNTF) over an 18-month period [An encapsulated cell technology implant, h period . Phase tdrug-eluting contact lenses reveal them as beneficial alternatives to anti-glaucoma drops and gels [Finally, the recent developments in and gels . ConsistDrug delivery to the brain is limited primarily due to the highly selective permeability of the blood–brain barrier. IDDSs are promising devices allowing drugs to bypass this limitation. Currently, the primary field of IDDSs intracerebral applications is the treatment of advanced malignant glial tumors.“Gliadel”) that have been approved by FDA to treat malignant gliomas [There are only four drugs  and one IDDSs  were coated with permanent polymers delivering antiproliferative drugs over a period of 90–180 days(paclitaxel (cytostatic), sirolimus, everolimus and zotarolimus (mTOR inhibitors)) ) . RestenoOrsiro, Synergy) were based on the bioresorbable polymer coatings of metal stents and delivered sirolimus, everolimus, and biolimus for 30–180 days. Sadly, the prevalence of restenosis was still 5–10%, which could be explained by the effects of both antiproliferative drugs and products of polymer degradation. The walls of the reperfused vessels were morphologically studied and showed delayed intimal proliferation and focused on chronic inflammatory reaction. Another complication of these stents was the development of neoatherosclerosis [The second generation of vascular IDDS  approved IDDSs includes subcutaneous, ocular, and intracerebral solid implants/inserts, as well as various pumps and drug-eluting stents see . The sumThe non-biodegradable nature of the majority of the currently commercially available IDDSs implies inevitable FBRs. However, the direct pieces of evidence indicating the fibrotic complications and excessive tissue growth at the implantation sites are relatively commonly reported only for subcutaneous inserts and for infusion pumps. At the same time, these types of adverse reactions are uncommon for ocular implants and drug-eluting stents. The complications linked to infections and the local effects of the eluted drugs are mostly non-specific to the IDDS itself. Additionally, the information available so far does not allow “blaming” of any specific vehicle material applied in IDDSs’ construction in the observed adverse responses .Key factors of FBR to the clinically used IDDS. The analysis of the data presented in this review allowed us to nominate a leading triad of the factors that significantly contribute to the FBR-related complications associated with the use of current IDDSs. Our analysis shows that this triad includes:(1)Implantation site (the immune reactivity of the host tissue);(2)The volume of the implant ;(3)The drug load of IDDS .One of the most unexpected findings emerging from the analysis of the current clinical landscape of IDDSs is that the engineering aspects of the modern IDDSs are addressed much better than the biology of FBR. It seems that there is a very good understanding of the efficient ways to create good implantable devices for drug delivery, and these principles can be re-used in new IDDSs with high chances of success. However, the tissue response such as  FBR still remains a challenging target. It certainly can be controlled further. This requires more research but also allows more room for improvement.We currently view IDDSs not as a separate medical technology but rather as a strong bioengineering trend with emerging applications in diverse clinical fields. However, this trend meets many sides of a universal host tissue response, the FBR. The analysis of the complications associated with different kinds of implantable systems indicates that many of the current challenges have non-specific solutions that will be implemented in systems developed in the near future.The history of the subcutaneous implantations of IDDSs in the upper-arm area proves that the minimization of the quantity of the system components and their sizes can be efficient for the long-term satisfactory patient experience. Moreover, the transition from Norplant, with six silicone rods, to Implanon, with one EVA rod, was better met by both patients and physicians because of the reduced volume of surgical procedures. Reports about inflammatory complications associated with subcutaneous implants are now scarce and indicate rare individual adverse FBR responses. The most discussed complication of upper-arm IDDS implantations is their possible migration towards the forearm area, which must be regularly monitored by the patient. The addition of the imaging tracer compound has not resulted in significant progress due to the problem of the device’s under-skin mobility. The current major trend in this field is the development of one-rod systems with reliable fixation of the implant in the tissue and predictable pharmacokinetics of the drug. This challenge, possibly, has two general solutions: (1) to create constant semi-permeable skin pockets for implants, or (2) to modulate FBR to control the implant’s position and functionality. From the observations of IDDSs used for intrathecal drug delivery (where the reservoir parts are placed in the skin pocket), it seems that the first approach can result in the increased incidence of missed injections and cosmetic defects.Implantable pumps’ applications reveal another, partially surprising, observation. First of all, only a small number of these IDDSs obtained FDA approval. The initial enthusiasm and expectations that any drug can become more efficient if it is delivered locally by a well-controlled mechanical pump soon met with the disappointment of the medical community following a massive promotion of a relatively raw technology. It took 25 years after the Infusaid pump became commercially available to reveal the source of the catheter-associated neurological sequelae. Implantable pumps did not become the standard of care for chemotherapy or diabetes treatment, but they found their niche in pain and spasticity management, where intrathecal delivery is crucial. Currently, we observe very careful and safe evolution of this technology in extension of application areas, the miniaturization of pump sizes and procedure protocols for drug refills, and doctor–patient dynamic dosage control.Intraocular implant technology has changed dramatically over the last 15 years. Earlier models demanded surgical fixation and removal, causing complications including fibrotic encapsulation. The most recent approach to make the IDDS so small that they can be delivered with a syringe paid off because of the closed space of vitreous body and immune privilege of the eye. This allows ophthalmologists to implant several IDDS; however, there is a need to control their possible migration to the anterior chamber of the eye.The only FDA-approved intracranial IDDS is Gliadel wafers. While there was no significant alteration of the technology, the adaptation of procedure protocols allowed the prolonged overall survival of patients with high-grade gliomas from 2 to 10 more months of life. This case clearly presents that IDDS technology is not about the search for panacea but about gaining maximum results from the medicines we have at hand.Cardiovascular surgery has the richest selection of IDDS. Unlike all implants mentioned above, drug-eluting stents were specifically designed for better biointegration with the blood vessel walls and serum proteins. These stents do not cancel the proliferation of neointima or stop angiogenesis but slow this process through the specific targeting of the mTOR pathway, which allows for the preservation of the required diameter of the lumen. In the next several years, we expect to observe how fully bioresorbable stents will take their place in the field. It is important, however, to continue monitoring possible defects and subsequent hemorrhages that can occur to semi-resorbed stents from mechanical stress. In addition, lessons learnt from the application of cardiovascular IDDSs point out the positive role of combining the materials with different properties, the usage of advanced polymer compositions, and mild and precise suppression of the FBR at the proliferation stage.Finally, it is important to remember that the IDDS–tissue interactions have two contributors. With the enormous potential of bioengineering to control the implant side, it is also very important to consider the local tissue contexts of the implantation site. The well-vascularized and immune active organs, such as skin and brain, may require more precise control of FBR, including the initial stages of acute inflammation to ensure the required level of cells’ adhesion to the device and its eventual integration. The vascular niche of the drug-eluting stents as well as the vitreous matter surrounding the ocular IDDSs are intrinsically inflammatory due to the disease that required treatment, while, certainly, they provide less extensive exposure to certain cell populations. Possibly, other drugs able to control local tissue proliferation at the implantation site  may enter the scene. Our own recent studies have demonstrated a possibility of re-purposing pirfenidone for FBR-controlling IDDS . Taken tIDDSs represent a very attractive therapeutic strategy that bypasses the limitations of the conventional drug administration routes. This technology contributes to the introduction of new bioactive substances to the clinical practice and helps to repurpose established medicines. It brings together physicians, researchers, engineers, industry companies, and venture business communities that have the energy and intentions to improve the quality of the patients’ lives and enhance the efficiency of treatment outcomes. The avalanche of clinical trials for the new IDDSs is anticipated to occur in the near future. However, we encourage clinicians and researchers to pay their attention to the lessons pointing at the importance of implant–tissue biointegration, which became clear during the formation of the current clinical landscape of IDDS technology. Most importantly, the experience of clinical application of IDDSs indicates the need to concentrate the efforts on the control of FBR via the biological mechanisms rather than by further increasing of the complexity of the engineering solutions. We believe that the current review has thrown some light on this important problem.


Editorial on the Research TopicOne- and Two-Dimensional Nanostructures for Drug Delivery ApplicationsSite specific and targeted delivery of therapeutic agents to specific body parts for the treatment of diseases is essential to increase drug efficacy and reduce toxic side effects. Employing specially designed nanoparticles as carriers of these therapeutic agents has proven effective for transporting drugs to the target and releasing when needed. Targeting is achieved by binding ligands to the nanoparticle surface to increase their uptake by the cells, enabling the targeting of different cells by changing the ligands used. Triggered release, which allows for drug release only at the target site, is achieved by using functional materials as the carrier particles that respond to changes in the target environment. Spherical nanoparticles are the most widely used nanostructures due to their relatively simple synthesis. However, spherical nanoparticles suffer from low drug loading capacities. Compared to zero dimensional nanoparticles , one-dimensional nanostructures  have the advantages of high loading capacities and enhanced targeting due to their high surface to volume ratios. However, compared to spherical nanoparticles, one-dimensional nanostructures have been explored less as drug carriers, which can be attributed to their relatively challenging fabrication methods.Longer circulation times, in addition to the alignment or tumbling of one-dimensional nanostructures during transport in the body, strongly impact the performance of these nanostructures as drug carriers. Although there are studies modeling the diffusion of these nanostructures in blood vessels, papers addressing the effect of their shape on the migration and transport mechanisms are limited. Furthermore, the effect of their shape on the uptake efficiency by the cells needs to be further explored. Similarly, two dimensional nanostructures have gained less attention compared to zero dimensional nanoparticles due to challenges in fabrication and severe immune response. However, recent studies have shown their significant benefits for particular treatments, such as multi-responsive control mechanisms and higher drug loading capacities. Therefore, this Research Topic invited researchers to contribute studies on recent advances in one- and two-dimensional nanostructures for drug delivery. We have collected three original research articles and one review, as reported below.Yeniyurt et al. in their publication present the functionalization of single-wall carbon nanotubes (SWNTs) with various Fmoc-amino acid-bearing polyethylene glycol (PEG) chains of different length by a non-covalent method. First, molecular dynamics studies allowed suitable Fmoc-amino acids to be selected for an effective surface coating of SWNTs; afterwards, in experimental studies the Fmoc-amino acids were associated to SWNTs and these carriers were then characterized. The effect of modified SWNTs on the viability of human dermal fibroblast cells was also evaluated.Balmori et al. address the important issue of protein corona formation on the surface of the nanoparticles used for cell-targeted delivery and investigated the effects of nanoparticle properties on the protein corona. The proteins adsorbing on the nanoparticle surfaces in the blood stream form a protein coating on the surface affecting the overall efficacy of the nanoparticles as cell targeting delivery devices. For this reason, a clear understanding of the interaction between the proteins and the nanoparticle surface is critical. Balmori et al. used Isothermal Titration Calorimetry (ITC) in combination with Nano Differential Scanning Calorimetry to study the effects of nanoparticle roughness and porosity on the adsorption of proteins to the particle surface. With the help of these two techniques, they could provide further explanation to the formation of hard protein corona formation inside the pores of the nanoparticles.In another study, Khlyustova and Yang in their paper describe the kinetics of deposition of poly (4-aminostyrene) (PAS) thin films by initiated chemical vapor deposition (iCVD). PAS thin films can be used as 2D-nanomaterials for drug delivery, since the primary amine functional groups can react with biomolecules that enable targeted delivery or biocompatibility. In addition, the iCVD technique allows to deposite PAS thin films uniformly over drugs enabling their effective encapsulation. Understanding kinetics of PAS polymerization in iCVD is crucial for such deployments, because drug release kinetics in thin-film encapsulation have been shown to be determined by the film thickness. The authors apply a dual-regime polymerization model to explain the growth rate as a function of fractional saturation pressure of the monomer: they demonstrate that at the critical fractional saturation pressure value of 0.2, a transition from quadratic dependence to linear dependence takes place.Gleason reviews the applications of polymeric nanolayers grown by iCVD for the controlled release of pharmaceuticals and other molecules. Starting from the description of the composition and the design of different iCVD polymers, the author describes the physico-chemical properties that they should possess for controlled release applications. The iCVD polymerization process is described in details and the characterization techniques are also reported. Finally, the paper reviews the wide range of structures that have been encapsulated by iCVD for controlled release of drugs, such as membranes, micro- and nanoparticles, fibers, textiles and porous media.One- and Two-Dimensional Nanostructures for Drug Delivery Applications” will contribute to the research field of drug delivery and increase the interest on one- and two-dimensional nanostructures as drug carrier systems.In summary, this Research Topic covers recent advances in the field of one- and two-dimensional nanostructures for drug delivery, providing new insights into effects of nanostructures on release kinetics and immune response to these nanostructures. The editors hope that the Research Topic “


Biaxial tensile testing methods using cruciform specimens have been developed in the last few decades for the determination of forming limit diagrams (FLDs) and fracture forming limit diagrams (FFLDs) for sheet metals. One of the difficulties associated with this test geometry is the lack of a widely accepted method to determine the necking and fracture strains which are necessary to construct these diagrams. In this study, a novel spatio-temporal method has been proposed for the determination of necking and fracture strains. In the method, two rectangular zones: the base zone (BZ) and the reference zone (RZ) are selected at the location where fracture initiates. The zone RZ includes the zone BZ and both zones have the same side length in the direction parallel to the necking band but different side length in the perpendicular direction. By plotting the thickness reduction within RZ against that in BZ, the onset of localised necking can be determined by finding the intersection of the two straight lines fitted separately using the data in the initial and final stages of deformation. The corresponding limit strains are then determined using the strains within the zone BZ. The method has been successfully applied to uniaxial tensile tests on AA6082 and boron steel dog-bone specimens, and to equi-biaxial tensile tests on AA5754 cruciform specimens.• Compared to widely used existing methods, the novel spatio-temporal method has greater simplicity, stability and accuracy with regard to the determination of localised necking strains.• The spatio-temporal method has good potential to become a standard method for the determination of limit strains for sheet metals.  Specifications Table Formability evaluation for sheet metals is essential for the development and optimisation of advanced forming technologies, especially for those at elevated temperatures (e.g. hot stamping). Forming limit diagrams (FLDs) and fracture forming limit diagrams (FFLDs) have been widely used as effective tools for the evaluation of formability. In FLDs, the limit strains at the onset of localised necking are presented in the space of major and minor strains, while in FFLDs, the limit strains at fracture are plotted on the same axes. Usually, FLDs are determined by using the Nakajima tests, and the limit strains at the onset of localised necking in the tested samples are measured using the cross-section method, as described in the international standard ISO 12004:2 2008 Biaxial tensile testing using cruciform specimens has great potential for the determination of FLDs and FFLDs under complex forming conditions. For this purpose, Lin et al. A spatio-temporal method has been proposed and developed for the determination of limit strains at the onset of localised necking and fracture for sheet metals. In this method, two rectangular zones, named the base zone (BZ) and the reference zone (RZ), are selected at the location where fracture initiates in the tested samples, as shown in The theoretical basis of the spatio-temporal method for the determination of onset of localised necking can be demonstrated by analysing the relationship between the average thickness reductions within the zones RZ and BZ. In the initial stages of deformation, variations in the thickness reduction within the two zones are equal because the deformation is homogeneous and thus one hasAfter the onset of localised necking, deformation mainly occurs within the necking band and the thickness reduction variation within the zone RZ-I  method in the standard ISO 12004–2:2008 The spatio-temporal method has also been applied to equi-biaxial tensile tests on 1.5 mm thick AA5754 cruciform specimens. The experimental details including setup and tensile speed have been given in Ref. In order to demonstrate the repeatability of the spatio-temporal method with respect to the limit strain determination, the uniaxial test  on boronThe equi-biaxial test  on AA575A spatio-temporal method has been developed for determination of necking and fracture strains, which are necessary to construct forming limit diagrams and fracture forming limit diagrams. In this method, the onset of localised necking is determined by analysing average thickness reduction within preselected zones around the location where fracture initiates. It has been validated that the method has stable ability to determine limit strains in either uniaxial tests using dog-bone specimens, or biaxial tests using cruciform specimens. Moreover, in the uniaxial tests, the method can reproduce results from the cross-section method which has been widely accepted. The method can be easily reproduced by following the procedure and recommended parameters.Compared with the cross-section method in the standard ISO 12004:2, the spatio-temporal method can be applied to cruciform specimens which usually have a relatively small gauge area. Furthermore, using the spatio-temporal method enables the determination of the limit strains at onset of necking and fracture in one process, which are necessary to construct the FLDs and FFLDs, respectively.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Intensification of pig farming increases the profitability of farms but also leads to greater local environmental pressure from manure nutrient loss. There are various technologies for pig manure processing. Each technological solution provides specific nutrient content in the end-products . The methodology was developed for choosing proper technological solutions for pig farms with due account for the whole variety of combinations of production and natural and climatic conditions. It includes an accurate accounting for nitrogen and phosphorus flows in the manure processing and utilisation chain and considers the limiting factors of the farm and the end-product customer requirements. The methodology was applied to a large-scale pig farm in the Leningrad Region. The best suitable processing technology was identified demonstrating the total amount of nitrogen and phosphorus in the end-products of 278.94 t per year. The developed methodology can serve as an effective tool to reduce manure nutrient loss and mitigate the adverse effect on the rural environment.The current trend towards larger pig farms increases their profitability but might harm animal welfare and the environment. More efficient pig manure management is a way to address this challenge. Available manure handling and utilisation systems may feature from 8 to 50% nutrient loss to the environment. Therefore, the proper choice of technological solutions is of high priority. In regard, the study developed a methodology including calculation, analysis and modelling techniques for the accurate accounting of manure amount, its fractions and their nitrogen and phosphorus content for different processing technologies with due regard to the limiting factors of the farm and the end-product consumer requirements. The methodology was applied to justify the best suitable processing technology for a large-scale pig farm in the Leningrad Region with 17,800 heads of pigs and 54,750 t of pig manure per year. The selected technology included manure separation into fractions, aeration and secondary sedimentation of the liquid fraction and passive composting of the solid fraction. It demonstrated the total amount of nitrogen (N) and phosphorus (P) in the end-products of 278.94 t per year, with the 26% total nutrients loss on all technological stages, and specific capital and operating costs of USD 55.5 per ton of manure produced. The methodology was tested by comparing the calculated data and the data from previous surveys of 15 pig farms in Russia. The differences between the values were found from 0.9 to 12.5% in mass; 2.8 to 13.9% in N content and 1.7 to 9.9% in P content. The developed methodology supports planning the production of the manure-based end-products in a given amount and with target nutrient content, depending on different processing technological solutions, achieving both economic and environmental goals. Intensification of pig husbandry raises the farms’ profitability, but it also entails a greater load on the local environment and may adversely affect animal welfare. The analysis revealed that nearly all intensive pig farms in Russia, especially large-scale pig rearing complexes, require their operation to be optimally improved in terms of economic profitability and minimal impact on the environment . Manure Manure nutrients  are considered the main source of environmental pollution, on the one hand, but they maintain soil fertility, on the other. However, currently, 47% of pig farms in the country have neither enough land to apply all organic fertilisers produced nor any arrangements with crop growing farms on fertiliser export ,4. In thResearchers in different countries offer their solutions to the problem of how to make intensive pig farming more environmentally sound. These solutions include, among others, the manure nutrients management system based on the mass balance method ,6,7,8, eThe accurate nutrient accounting requires calculation methods of the quantity and quality of pig manure and resulting organic fertilisers and other by-products ,18,19,20A technological solution for pig manure processing is selected based on the technical and land resources of the farm and the end-product requirements of customers. However, any technological solution is expected to apply all tools to minimize emissions in the production process ,22,23. POne way to optimise the nutrient flow is to separate the pig manure into solid and liquid fractions ,26,27 anIt is important to consider the nutrients produced both at the farm level and in larger agroecosystems at a district or region level ,34,35. IHowever, if a large-scale pig farm has no agricultural land, the use of pig manure as a liquid organic fertiliser is uneconomical. It is worth considering other end-product options. In a concentrated form, they might be more cost-effective organic fertilisers. While the nutrient-poor cleaned water can be reused at the farm.The scientific hypothesis of the work is that the environmental sustainability of a pig farm is achieved, among others, by creating a balanced system of the effective transfer (flow) of nutrients (nitrogen and phosphorus) from the pig manure into the end-products. An effective tool in this respect is to choose the technological solutions that consider the characteristics of both the source material and the target products .The study objective was to assess the environmental and economic performance of a large-scale intensive pig farm located in the Baltic Sea catchment area. For this purpose, a special tool (methodology) was designed to support the choice of such a technological solution to manure processing that would be best suited in terms of available resources, the end-product requirements and environmental effect.The methodology will include the calculation techniques for the quantity and quality of the fresh manure and the end-products (in terms of the customer needs), the computational analysis of various technological solutions to converting manure into the end-products, the modelling of mass and nutrient distribution and the estimation of relevant costs.Following the study aim, intensive pig farming in the Russian part of the Baltic Sea Region was considered using a systemic approach.A present-day large-scale pig farm, as a rule, has several production sites and may cover a vast area since its numerous production facilities and fields are located at a substantial distance from each other.Therefore, such a farm may be regarded as a separate integrated agroecosystem. Its sustainability is of great importance for both the farm itself and the environment.Such a farm has the sources of point pollution , non-point pollution (fields with applied organic fertilisers) and combined pollution  .A closed-type large-scale pig farm is a self-sufficient “mono-system” where production and processing take place right on the farm without involving external players. Its characteristic features are sufficient capacity of pig manure processing sites and storages; enough agricultural land within a cost-effective transportation distance to apply all organic fertilisers produced; the owned feed mill and meat-processing facility; and cultivation of crops for the own use for feed and sale, and direct selling of pork and by-products.A combined-type large-scale pig farm is a “mono-system” with minimal involvement of external players. In contrast to the closed-type farm, it does not have a feed mill and purchases the feeds. Available agricultural land is not enough to apply all organic fertilisers produced. Therefore, such a farm rents some land for fertiliser application that sometimes leads to uneconomic transportation distances.An open-type pig farm is a “mono-system” involving many external players. It differs from the combined-type farm in that the pig manure processing and organic fertiliser field application are passed to third-party organisations.Long-term experience of research associated with technological solutions for pig farms allows dividing them into three types.A comprehensive analysis of available technological solutions to pig manure handling allows dividing them by the obtained end-products into three groups: single-product technological solutions, two-product technological solutions and multi-product technological solutions.The output of single-product technological solutions is the solid or liquid organic fertilisers, with the target being the maximum saving of nutrients (minimum nutrients loss).The output of two-product technological solutions is an organic fertiliser (solid or liquid) and water (effluent) that can be used for additional fertilisation of crops or process needs. After additional biological treatment, it can be discharged into the open water bodies if its chemical composition complies with established standards. The target of such technological solutions is to concentrate nutrients in the organic fertiliser to reach the required quality of the cleaned water.The output of multi-product technological solutions is organic fertilisers (solid or liquid), cleaned water and other end-products: biogas, electric energy, methane tank effluent and others. The target of such technological solutions is the redistribution of nutrients depending on the end-product requirements .Characteristics of raw manure to be processed and the end-products  are considered separately for each technology.The study integrated several previously worked out methods, including those of other researchers, in the methodology under development. The methodology is understood as a body of methods, approaches and principles employed to reach the research objectives.The study of two-product and multi-product technological solutions included the survey of design documents, scientific reports and publications for 1970–1990. Those years, all large pig rearing complexes under construction in the Russian Federation and Eastern Europe widely introduced the technologies for multi-stage processing of the liquid fraction of pig manure and fermentation of the solid fraction. The obtained data were used to calculate the basic coefficients in the models describing technological processes. Due to the recent changes in animal diets and manure removal solutions, additional laboratory and farm-scale studies were performed to update the basic coefficients.t-test. The coefficients values reflect the influence degree of factors on the dependent variable.Mathematical models were obtained by the method of least squares of the regression equation. The resulting models and coefficients are statistically significant. The statistical significance of the models was tested by Fisher’s criterion and determination coefficient. The statistical significance of the coefficients was checked by Student’s The study applied theoretical methods of functional and structural analysis, decomposition of technological processes, expert evaluation method, mathematical methods of optimisation, theoretical modelling methods and laboratory experiments on pig manure from selected pig farms.The experimental data were analysed in Microsoft Excel 2016 and Statgraphics Centurion 16.1.11 software packages.The calculations apply the mass balance method ,6,7,8. TThe data received on the pig manure amount and its nutrient (nitrogen and phosphorus) content is used to calculate the quantity and quality of resulting organic fertilisers with due account for specific features of processing technologies in place in the Russian part of the Baltic Sea Region. Reasonable nutrient loss factors are used for each stage of manure and fertiliser handling—transportation of manure from livestock houses to either processing, temporary accumulation place, or both, transportation of end-products and organic fertiliser, use of end-products, and application of organic fertilisers, with the specific values of the factors being found in ,8,18,22.The procedure includes the models based on the principle of efficient use of nitrogen and phosphorus in the farm operation chain ,40.NUE) is calculated asNitrogen use efficiency (Phosphorus use efficiency (PUE) is calculated in a similar way.The procedure also applies the mathematical models for calculating the concentration of nutrient content in organic fertilisers to achieve the target quality of cleaned water. They contain the coefficients characterising the operating modes and process parameters of equipment and calculated coefficients characterising required quantity and quality of each type of end-products ,42,43,44The method involves the use of mathematical models, which describe the most common and emerging technological solutions for pig manure processing: long-term storing (maturing), passive and active composting, fermentation in drum and chamber fermenters and multi-stage processing with biological treatment ,48,49,50The calculations associated with well-researched technological solutions and processes used the known formulas and dependencies ,44,45,47For the under-studied technological solutions, the experimental studies were performed to obtain the coefficients required in the calculations.The subject of a separate laboratory-scale experiment was three-stage processing of the liquid fraction of pig manure. Stage 1 was sedimentation in a vertical settling tank. Stage 2 was aeration of the supernatant (clear water) followed by sedimentation in a settling tank. Stage 3 was the long-term storing (maturing) of the mixture of sludge from Stage 1 and excess activated sludge from stage 2 .−1−1−1−1−1The results of the experiment were used to establish dependencies between the amount of cleaned water, its total nitrogen and total phosphorus content and the duration of technological processes (Formulas (2)–(5)):−1−1−1.A multilevel model of the technology for multi-stage processing of the liquid fraction of pig manure is presented by dependencies (6):These dependencies helped select an optimal set of technological solutions for the multi-stage processing of pig manure to achieve the required quality and quantity of the end-product—cleaned water.The established mathematical dependencies provide a way to estimate the adaptability of the technology to the farm resources and consumer requirements; to identify the main operation modes and the possible variation boundaries of the coefficients characterising the operation modes in the practical technology implementation.The experimental data were analysed in Statgraphics Centurion 16.1.11. The correlation coefficient was 0.98, the determination coefficient was 96%.The subject of another separate experimental study was the fermentation of the solid fraction of pig manure in a laboratory drum fermenter with the solid organic fertiliser as the end-product ,53.The manure was separated into fractions on a pig farm by a screw separator. Before loading the solid fraction into the laboratory fermenter, the mass was measured and samples were taken to determine the moisture, total nitrogen and total phosphorus content. The fermentation lasted for 7 days. The fermenter was installed on a strain gauge balance. The measurements of the mass and the sampling took place every day. The experiment was performed in three replications. The experimental data were analysed in Statgraphics Centurion 16.1.11.The results of the experiment were used to establish dependencies between the mass of resulting organic fertiliser, its total nitrogen and phosphorus content and the fermentation time:−1.The correlation coefficient was 0.98.−1.The correlation coefficient was 0.98, the determination coefficient was 97.6%.The determination coefficient was 99.8%.The study applied the mathematical apparatus of the transportation problem of organic fertiliser transfer from supplying farms to consuming farms. The target function was the transportation cost of one ton of total nitrogen .The optimal nutrient balance in the agroecosystem means that nutrients return to the production cycle with minimal loss to the environment. Step 1 is to acquire the initial data on the pig farm. Step 2 is to calculate the quantity and quality of pig manure as a raw material with due account for the technologies in place. Step 3 is to identify the applicability of different technological solutions depending on natural and climatic conditions and the specific performance characteristics of the pig farm. Step 4 is to make a graphic representation of correlations between applicable technological solutions within the pig manure processing technology into the end-products. Step 5 is to calculate the quantity and quality of the end-products by mathematical models and to substantiate the optimal for introduction technology. Step 6 is to estimate the application options of remained organic fertiliser and to calculate the economic efficiency. The information obtained about the suggested technology can be used for a comprehensive agro-environmental and economic assessment, including the future estimation of greenhouse gas emissions and the possibility of using such a resource as clean water (step 7).The established system of methods provides a way to assess the level of environmental compliance of pig farms and design an effective set of machines and technologies for the environmental sustainability of these farms as agroecosystems.A comprehensive analysis of existing pig manure handling solutions with the use of the calculated coefficients made it possible to draw up a general procedure to obtain the end-products from pig manure fractions with specified nutrients content by the following methods: natural biological processing; a combination of physical actions accompanied by natural biological processes in separated pig manure fractions; a combination of physical actions and specially organised biological processes; and a combination of physical and chemical actions and organised biological processes .The developed methodology was tested on the data from a selected full-cycle pig farm with a total pig stock of 17,800 heads and annual pig manure output of 54,750 t located in the Leningrad Region, i.e., in the Russian part of the Baltic Sea catchment area. Its emissions and diffuse inputs of nutrients were of special importance to be estimated in terms of marine environment protection [To analyse the substantial amount of data, we designed a computer-aided programme for assessing and selecting adaptive technologies for pig manure processing, which was approved by the State Certification Authority.The developed methodology was applied to a full-cycle combined-type large pig farm with a total pig stock of 17,800 heads and annual pig manure output of 54,750 tons located in the Leningrad Region.The animals were housed on a partially slatted floor without bedding except for the suckling piglets. The pig manure was removed from the livestock houses by the gravity system of batch type.At that time, the pig farm separated manure into fractions, with the long-term storing (maturing) of the liquid fraction and passive composting of the solid fraction. The resulting solid organic fertilisers were applied to the owned agricultural land in the correct doses. The resulting liquid organic fertilisers were applied to the owned agricultural land in significantly higher doses.The main limiting factor of the pig farm was the lack of sufficient agricultural land for the application of organic fertilisers. At least 1800 hectares of agricultural land were required to apply all the fertiliser obtained. For cost-effective fertilisation, the distance between the fields and the farm should not exceed 15 km. However, on the farm only 1280 hectares at a distance of above 15 km from the manure processing site were available.The study objective was to apply the developed methodology to identify how to produce more concentrated fertiliser for transportation in smaller volumes over long distances.−1 of pig manure with 93.4% moisture content, 795 kg of total nitrogen content and 247 kg of total phosphorus content.The first step was to calculate the daily pig manure output, its moisture content and the initial content of total nitrogen and total phosphorus in the amount of pig manure transferred to processing by the values from regulatory documents. The calculation results were 150 t dTechnology 1—long-term storing (maturing) (LTS (M));Technology 2—separation into fractions (SF) + long-term storing (maturing) (LTS (M)) + passive composting (PC);Technology 3—separation into fractions (SF) + sedimentation in tanks with stop-logs and treatment in biological ponds (S + BP) + passive composting (PC);Technology 4—separation into fractions (SF) + aeration and flocculation (A + F) + passive composting (PC);Technology 5—separation into fractions (SF) + aeration and coagulation (A + C) + passive composting (PC);Technology 6—separation into fractions (SF) + aeration and sedimentation in batch-type settling tanks (A + S) + passive composting (PC).The second step was to analyse the natural and climatic conditions, soil properties in the location of the selected pig farm and the technical possibility (land availability) to introduce the possible technological solutions for obtaining the manure-based end-products see . Six tecThe economic and environmental indicators were calculated for the selected technologies .The amounts of nitrogen and phosphorus in the end-products calculated for the above six technologies are the nutrients saved in the end-products (organic fertilisers) taking into account the losses at each technological stage—manure processing, temporary accumulation of end-products and transportation of end-products to their application place.The area required to apply the calculated amount of fertiliser nutrients was compared with the area available on the farm.The third step was to choose the optimal technology by the method of multi-criteria Pareto optimisation  Table 2Table 2. According to the ecological indicator, technology 1—long-term storing (maturing) was the most optimal for the pilot pig farm. However, the available land was not enough for its implementation. The organic fertilisers needed to be transported at disadvantageous distances, resulting in a higher economic indicator.The developed computer-aided programme found technology 6 (separation into fractions + aeration and sedimentation in batch-type settling tanks + passive composting) the most suited for the pilot pig farm following the selected criteria and resources .The programme also identified the equipment required for this technology, calculated its number, dimensions and maintenance conditions  Table 3Table 3. A balance diagram of the selected technology was created  to deterTo verify the developed methodology, the calculated data on 15 large pig farms in Russia, with seven of them being located in the Russian part of the Baltic Sea catchment area, were compared with the data from previous surveys of these farms when elaborating technological regulations for manure management.To ensure data reliability, the initial data were acquired and analysed through a questionnaire survey of farms, examining the technological maps and technological processes as well as the protocols of laboratory analyses supplied by the accredited laboratories. The adequacy of acquired data was verified by organising field surveys of 8 out of 15 pig farms, including the sampling and sample analysis with the methods commonly used in Russia and Europe.The obtained experimental data were statistically analysed. The mean values and standard deviations (SD) were calculated. The average experimental values for all pig farms fell in the interval of standard deviations. Therefore, the data were considered reliable .Pig farm 1 is located in the Kaliningrad Region; pig farms 2–7 are located in the Leningrad Region; pig farm 8 is located in the Pskov Region; pig farms 9 and 10 are located in the Republic of Buryatia; pig farms 11–13 are located in the Krasnoyarsk Territory; pig farm 14 is located in the Tomsk Region and pig farm 15 is located in the Kaluga Region.The pig housing systems and technologies for manure removal and processing into end products were considered. The developed methodology was applied in calculations .From These differences are not significant in the aggregated assessment of the large-scale intensive pig farms. Accordingly, the developed methodology can be used for an aggregated assessment of the end products based on pig manure both at the farm, district or region level.The intensification of pig farming is a current worldwide trend. The major challenge for intensive large-scale pig farms in Russia is to process and utilise the large amounts of manure with minimal environmental pressure (manure nutrient loss) under limited owned agricultural land and too-long transportation distances. An effective way is to choose the relevant technological manure processing solutions that would provide the balanced N and P transfer (flow) from the raw manure to the target end-products with specified characteristics  We designed a special tool (methodology) to support this choice through calculation of quantity and quality of the fresh manure and the end-products (in terms of the customer needs), the computational analysis of applicable technological solutions, the modelling of mass and nutrient distribution and the estimation of relevant costs.The methodology is the first basic step in creating a comprehensive system of designing and effective management of pig farms based on available resources, maximum nutrient saving, limiting factors, economic and environmental considerations. This instrument is also expected to support the decision-making on the regional level on the subsidizing of farms in the transition to Best Available Techniques and the environmental permitting process. In the future, it may be applied together with the terrain estimates to design new pig farms and forecast the possible adverse environmental effects at the farm, district or region level.A limitation of our methodology lies in some initial data acquisition for calculations—we used the literature data on soil characteristics when calculating the organic fertiliser application rates and the data provided by the pig farms on target yields. The required amount of nutrients was calculated from the nutrient removal with the yields. In the future, on-farm agrochemical soil surveys shall be provided.The methodology was developed with reference to a large-scale pig farm in the Russian part of the Baltic Sea catchment area. The best-suited manure processing technological solution was identified to provide the most efficient environmental and economic performance.The methodology was further tested on 15 large pig farms located in different climatic zones of Russia with their specific animal diets depending on the region, farm profile, feed base and production capacity. The calculated data based on the aggregated coefficients were compared with the verified data from previous surveys of these farms. According to the test results, the methodology can be applied to an aggregated assessment of the pig manure-based end products. However, more accurate calculations require additional examining of pig farms.The methodology integrated several methods, calculation and modelling techniques used by researchers in other countries.First of all, this is a digital tool based on the mass balance method, which considers the nutrient flow throughout the production chain—from feed to pig excrement and organic fertilisers ,6,7,57. We also applied the European calculation method through nitrogen use efficiency (NUE) ,8,18,22,Many researchers use the modelling to predict the pig manure evolution in terms of mass, dry matter and nutrient content and related gaseous emissions. However, they either consider the flows from fresh manure to compost ready for field application as  or from Our methodology suggests the optimal technological solutions based on the customer farm needs: crop farms need organic fertilisers; an industrial enterprise needs biogas; the landless pig farm needs to accumulate all the nutrients in a smaller volume of organic fertiliser for lower costs of long-distance transportation, etc.An idea of our methodology originating from the lack of agricultural land to consider other end products from pig manure with increased nutrient content and more feasible export to other farms (districts) resonates with other studies. In  the soliTo improve the pig manure management systems, the researchers suggest different approaches. The authors of  apply BeTo estimate the current pig manure management and suggest improvements, many researchers use the life cycle assessment (LCA) ,60,61,62In general, the designed methodology seems to match the European search for the most efficient use of manure nutrients through producing various end-products of manure processing.It also could support achieving some international goals of Russia, namely, to reduce the nutrients inputs into the Baltic Sea according to the target indicators specified by the HELCOM Baltic Sea Action Plan ,63. On tA methodology was developed integrating methods, procedures and models to assess the environmental performance of large intensive pig farms. It was applied to a full-cycle pig farm in the Leningrad Region (Russia). Six options of pig manure processing into the target end-products were selected in terms of natural, technical and economic conditions and the limiting factors of the farm. The best suitable technology was identified, with the economic indicator, i.e., the sum of specific capital and operational costs, being USD 55.5 per ton of pig manure and the ecological indicator being 278.94 t of total nitrogen and phosphorus in the end-products per year.The methodology was further tested by comparing the calculated data on 15 large pig farms in Russia with the verified data from previous surveys of these farms when elaborating technological regulations for manure management. The maximum difference between the calculated values and the actual data was 12.5% in the end-product amount, 13.9% in the end-product total nitrogen content and 9.9% in the end-product total phosphorus content. These differences are not significant in the aggregated assessment of large-scale intensive pig farms.The study results are expected to become the starting methodological point for the creation of advanced waste management systems in intensive pig farming through a comprehensive approach to producing different end-products by the most effective manure processing technology and with the least manure nutrient loss to the environment at the farm, district and regional level.