In recent years, virtual reality (VR) technologies have become widely used in clinical settings because they offer impressive opportunities for neurorehabilitation of different cognitive deficits. Specifically, virtual environments (VEs) have ideal characteristics for navigational training aimed at rehabilitating spatial memory. A systematic search, following PRISMA guidelines, was carried out to explore the current scenario in neurorehabilitation of spatial memory using virtual reality. The literature on this topic was queried, 5048 papers were screened, and 16 studies were included, covering patients presenting different neuropsychological diseases. Our findings highlight the potential of the navigational task in virtual environments (VEs) for enhancing navigation and orientation abilities in patients with spatial memory disorders. The results are promising and suggest that VR training can facilitate neurorehabilitation, promoting brain plasticity processes. An overview of how VR-based training has been implemented is crucial for using these tools in clinical settings. Hence, in the current manuscript, we have critically debated the structure and the length of training protocols, as well as a different type of exploration through VR devices with different degrees of immersion. Furthermore, we analyzed and highlighted the crucial role played by the selection of the assessment tools.  Two higFrom the search of both databases, we obtained 5048 articles, excluding duplicates. Title and abstract screening was carried out, and 24 articles passed to the full-text screening phase. Eight studies were excluded with reasons as follow: Not Controlled trial (= 1); Results of neuropsychological outcome for spatial memory not reported (= 3); Qualitative/descriptive study (= 2); Not neuropsychological rehabilitation for spatial memory (= 2).The review considered randomized control trials, nonrandomized control trials, intervention studies, and case-control studies in clinical patient populations with an overt spatial memory disorder. Studies on rehabilitation’s programs of navigation abilities with virtual reality (VR) devices in different population of patients (such as mild cognitive impairment (MCI), Alzheimer’s disease (AD), traumatic brain injury (TBI), multiple sclerosis (MS), stroke, cerebral palsy, epilepsy, incomplete cervical vertebro-spinal trauma, topographical disorientation disorders and neglect) were included. We only included studies in the English language and which satisfied strict criteria for eligibility for the review . The qualitative component also considered the type of VR navigational training and methodological design. We excluded articles which lacked necessary information for review in the full-text or the abstract. Reviews, meeting abstracts, proceedings, notes, case reports, letters to the editor, assessment protocols, editorials, and other editorial materials were also excluded. Retrospective studies were not included because the area of interest requires performing experiments. To assess the risk of bias, the reviewers followed the methods recommended by The Cochrane Collaboration Risk of Bias Tool  and the PRISMA guidelines were strictly followed; all titles and abstracts were screened according to the abovementioned inclusion criteria after removing the duplicates. Full texts of eligible articles were retrieved and assessed by two reviewers (J.M. and C.T.) for individual selection of papers to reduce the risk of bias and resolving disagreements through consensus as explained in Sixteen studies were analyzed to test the usefulness of rehabilitative interventions using virtual reality (VR) systems. However, growing interest in VR has led researchers to question how the characteristics of VR equipment and different aspects of the training tasks could influence the treatment outcomes, with particular regard to the results that reflect on the patient’s daily life in an ecological way. In our review, we aim at giving more awareness to the researchers and at guiding them in the selection of the most appropriate VR device to use. Considering the studies mentioned in this analysis, it could be possible to understand which is the most suitable program for the treatment of spatial memory disorders, in terms of the type of apparatus used and the training method. Furthermore, it is essential to understand which kind of patient will benefit from the intervention. To satisfy our aims and to facilitate the understanding, we considered the following clusters: (1) Authors; (2) Year; (3) Sample (N); (4) Sample characteristics; (5) Mean age; (6) VR Task; (7) Virtual Apparatus; (8) Neuropsychological assessment; (9) Primary Outcomes. Results are reported in Analysis of results in spatial navigation rehabilitation programs was carried out starting from the outcomes of 16 studies, taking into consideration the patient population, the VR apparatus (immersive/semi- or nonimmersive) and the type of training used. We analyzed clinical, methodological, and technical outcomes to cast research and clinical applications of VR in the context of VR navigational training. The results were argued in response to the following three questions. Among all the studies reviewed, the only one that used an immersive virtual reality (VR) system with a head-mounted display HMD Oculus Rift DK2, using the Unity 5 game engine, and a joypad is described in  . However, better performance can be achieved through immersive rather than semi-immersive systems by using head-mounted displays. This is explained by the greater sense of presence ,8, with The studies included in the current review are all focused on the rehabilitation of spatial memory and navigation abilities in different neurologic patients, including mild cognitive impairment (MCI), Alzheimer’s disease (AD), traumatic brain injury (TBI), multiple sclerosis (MS), stroke, cerebral palsy, epilepsy, incomplete cervical vertebro-spinal trauma, prosopagnosia with topographical disorientation disorders, and neglect. all patients have used virtual reality (VR) devices in different rehabilitation programs, and nobody had any difficulty in following the training. Overall, the emerging outcomes are positive, and they present improvements to different degrees. All of the mentioned studies had a control group ,57,59,61The protocol duration turned out to be an important variable for rehabilitation training outcomes , particularly in visuospatial , attention , and memory  domains and in the Mini-Mental State Examination (MMSE)  r = 0.474Results of the current review support the idea that virtual reality-based training improves orientation and navigation abilities in different neuropsychological disorders. Although the efficacy of treatment needs to be critically analyzed using a scientifically validated method, for this reason, the use of validated measures with adequate psychometric properties is fundamental. Over the past decade, evidence-based clinical guidelines have become a significant feature of healthcare services to improve the quality of care . Indeed,In the current review, we provided initial, positive results concerning the effect of virtual reality (VR) training on spatial memory rehabilitation, highlighting the potential of navigational tasks in virtual environments (VEs) to enhance navigation and orientation abilities in patients with spatial memory disorders. The rapid development and diffusion of VR technologies are amending the accessibility landscape of VR technology for the average consumer. Lower-cost VR systems such as the Oculus Rift, Go, Quest, and the HTC Vive are already issued on the market and have significantly reduced the cost barrier of VR hardware. Even lower-cost options are currently available using a smartphone, for example, Gear VR is compatible with specific Samsung phones, and both Google Cardboard and Google Daydream can be used with several smartphones. Although the review underlined encouraging results, current research in this topic has some limitations that researchers need to overcome. The current work is meant to provide methodological solutions for future studies. As the first result of the review, we have found clear improvements in spatial memory through the application of navigational tasks in VR. Both immersive and nonimmersive VR systems have shown appropriate enhancements for navigation and orientation abilities, underling the power of the navigational tasks proposed. Furthermore, the results have shown a transference of improvements from VR-based training to more general aspects of spatial cognition. However, the mode of exploration influences the spatial learning of a new environment. The active exploration has an essential role in the acquisition of spatial knowledge and it is characterized by five components: motor orders that determine the path of locomotion, proprioceptive and vestibular information for self-motion, allocation of attention to navigation-related features of the environment, and cognitive decisions about the direction during navigation and mental manipulation of spatial information . If immeAnother important element for the rehabilitation program is the structure and length of the training protocol. Some studies have already demonstrated that the use of visual cueing, such as a map as a navigational aid, improved the performance of users performing complex navigational tasks  and in pThe rehabilitation programs analyzed all focused on spatial memory and navigation abilities in different neurologic patients, including mild cognitive impairment (MCI), Alzheimer’s disease (AD), traumatic brain injury (TBI), stroke, epilepsy, prosopagnosia with topographical disorientation disorders, and neglect. An important aspect to keep in mind is the clear characterization of the sample of patients to whom the therapeutic procedure is provided. The severity and the qualitative characteristics of the mnestic deficit are extremely variable from subject to subject. Furthermore, memory disorders rarely occur in an isolated form, and are often accompanied by an impairment of other cognitive functions, such as attention, language, reasoning. In this view, the most effective protocols are the ones that target a specific pathology. The difficulty in finding homogeneous groups of patients is the reason why most of the experimental studies reported in the literature are based on the treatment of a single patient or a tiny group of patients. However, to monitor the improvements due to the treatment, the presence of an adequate control group is necessary to ensure that the observed improvement is not due to spontaneous recovery nor is the result of generic cognitive stimulation. In this view, the most reliable control condition is the repeated evaluation of the same patients receiving the experimental treatment, for example, through cross-over trials . AnotherThis review was subject to certain limitations. Extensive literature research was performed to deliver these findings; however, only two databased were queried. A second limitation of this review may be the selection of the keywords in the search strategy. In order to provide a considerable quantity of studies in the field, the authors decided to use wider keywords. However, the inclusion criteria reduced the focus to only studies according to the objectives of the review. A final limitation may be the fact that the discussion on duration and intensity of VR navigational training cannot draw firm conclusions due to the heterogeneity of interventions. Moreover, the difficulty of finding a homogeneous group of patients is the reason why most of the experimental studies reported in the literature are based on the treatment of a single patient or a tiny group of patients. Because our findings are positive, we hope that the literature could soon be enriched with clinical trials investigating VR’s effectiveness in more specific and larger clinical populations.In conclusion, the results of this systematic review demonstrate that all studies, although to varying degrees, suggest that patients improved their spatial memory following treatment. This result highlights the potential of navigational tasks performed in virtual environments (VEs) for enhancing navigation and orientation abilities in patients with spatial memory disorders. In neuroscience, researchers have long faced the challenge of conducting ecologically valid measurements of experimental variables while maintaining strict experimental control over visual displays. Virtual reality (VR) systems enable the researchers to design and consequently control dynamic, realistic, and immersive environments, while closely monitoring behavioral and physiological responses during experimentation ,73. In t


Danio rerio) is a valuable model for studying human physiology, disease, and toxicity. Additionally, the anatomical components of the zebrafish olfactory system are similar to those of other vertebrates, and they present a remarkable degree of regeneration and neuroplasticity, making it an ideal model for the study of regeneration, reorganization and repair mechanisms following olfactory toxicant exposure. In this review, we focus on (1) the anatomical, morphological, and functional organization of the olfactory system of zebrafish; (2) the adverse effects of olfactory toxicants and injury to the olfactory organ; and (3) remodeling and repair neuroplasticity mechanisms following injury and degeneration by olfactory toxicant exposure.The olfactory system, composed of the olfactory organs and the olfactory bulb, allows organisms to interact with their environment and through the detection of odor signals. Olfaction mediates behaviors pivotal for survival, such as feeding, mating, social behavior, and danger assessment. The olfactory organs are directly exposed to the milieu, and thus are particularly vulnerable to damage by environmental pollutants and toxicants, such as heavy metals, pesticides, and surfactants, among others. Given the widespread occurrence of olfactory toxicants, there is a pressing need to understand the effects of these harmful compounds on olfactory function. Zebrafish ( The olfactory system plays a pivotal role in animal survival since it detects and discriminates myriad odor signals and mediates important behaviors such as foraging, mating, social behavior, and assessing danger ,2. The sDanio rerio), a small freshwater teleost fish, has emerged as a valuable and relevant model for studying human physiology, disease, and toxicity. The major organs and tissues of zebrafish share molecular, anatomical, and physiological features with their mammalian counterparts, and it has been established that over 70% of zebrafish genes are shared with humans [It is well established that the functional and structural organization of the olfactory system, as well as olfactory transduction mechanisms, have remained evolutionary conserved in vertebrates ,11,12,13h humans ,15,16. AIn particular, zebrafish is an ideal model for studying the olfactory system, since its anatomical components are more accessible in this animal than other vertebrates. This makes the zebrafish olfactory system very amenable to experimental manipulations with less invasive techniques, increasing the rate of survival and recovery ,17,18,19Here, we will provide a comprehensive overview of (1) the anatomical, morphological, and functional organization of the olfactory system of zebrafish; (2) the adverse effects of neurotoxicants, chemical damage, and physical injury in the olfactory organ and olfactory bulb; and (3) remodeling and repair neuroplasticity mechanisms following toxicant exposure and injury to the olfactory organ. This review will serve as a foundation for the use of zebrafish as a useful and robust model for assessing the effects of olfactory toxicants and injury.The overall architecture and functional organization of the olfactory system of zebrafish is analogous to that of other vertebrates and has been described in detail. It is formed by two main structures: a pair of peripheral olfactory organs, or rosettes, located in the nasal cavity connected to the olfactory bulbs, which constitute the rostral-most forebrain region A 9,10,1,26,27,28The olfactory organ of zebrafish includes epithelium arranged in several lamellae that converge in a central non-sensory raphe, forming a bilaterally symmetrical, cup-shaped structure known as the rosette. Lamellae are composed of a continuous sensory area, found in the central and medial region of the rosettes, as well as a surrounding non-sensory epithelium located dorsally B 10,43,,43,44. TOSNs express a combination of discrete olfactory receptors (ORs), and groups of OSNs expressing the same olfactory receptors are positioned in defined zones within the rosette ,51,52. EORs belong to the G protein-coupled receptor (GPCR) superfamily and are further divided in three types: (1) olfactory receptors (ORs), the most abundant type, further divide into families and subfamilies ,44,59,60In sum, the anatomical and functional organization of the olfactory organ, with a defined topographic map of OSNs and their cognate olfactory receptors, creates chemotopical domains of olfactory processing that enhance the discrimination of thousands of individual odorants and their combination in a precise fashion.The olfactory bulb (OB) is a paired brain structure located in the most rostral region of the forebrain, connected to the olfactory organ by a short olfactory nerve; it constitutes the central relay of the olfactory system where olfactory information is processed and transmitted to telencephalic areas. The morphological organization and circuitry of the zebrafish OB are well understood and simpler than in mammals. The OB is organized in three diffuse laminae: (1) the olfactory nerve layer (ONL), formed by OSN axons ,54; (2) The OB receives sensory input from peripheral OSN axons that segregate to form discrete glomeruli. About 140 glomeruli have been identified, with 27 glomeruli being easily recognized. These are organized in nine clusters based on location, OR expression, and size and are designated as follows: dorsal glomerular (dG), dorso-lateral (dlG), lateral (lG), medio-dorsal (mdG), medio-anterior (maG), medio-posterior (mpG), ventro-anterior (vaG), ventro-posterior (vpG), and ventro-medial (vmG) ,31. AxonIt is well established that the glomerular topographical organization of the OB creates a spatial representation of olfactory input from the OSNs that is conserved between both bulbs. Ciliated and microvillous OSNs project to specific glomeruli in a mutually exclusive manner; therefore, no glomerulus is innervated by both types of OSNs C. CiliatTo summarize, the topographical map of OSNs expressing similar types of ORs in the olfactory sensory epithelium, followed by the clustered axonal projection of these OSNs in specifically located glomeruli, elicits the representation of different odorant categories in the olfactory bulb. This odor map allows glomeruli to process and enhance responses to specific odorants with exquisite precision. Moreover, the further organized odor-processing output pathways to higher brain centers permit eliciting of specific odor-mediated behavioral responses crucial for zebrafish survival, such as foraging, mating, and social behaviors.In a fast-changing environment, olfaction provides information regarding environmental conditions such as food availability, kinship, mating availability, danger signals, and death, among many others. Fish rely on these olfactory signals to rapidly adjust their behaviors in order to survive. Odors mediate pivotal survival behaviors in fish, including feeding, alarm, schooling, avoidance response, mating, and migration. Modification of these behaviors requires that the olfactory system responds to olfactory cues in a prompt and dynamic fashion. Zebrafish offers a favorable model for studying both the mechanisms that underlie odor-sensing and olfactory alterations, as well as their behavioral effects. Regardless of the molecular mechanism behind them, olfactory alterations can be functionally grouped into three categories: (1) anosmia, a complete loss of olfaction; (2) hyposmia, a reduced olfaction capacity; and 3) dysosmia, dysregulated olfactory processing . These a dysosmiaZebrafish initiate easily identifiable swimming patterns in response to diverse olfactory cues. General chemotaxis behavior includes attraction towards positive chemical cues  and avoidance to aversive chemical signals . Chemically mediated behaviors are characterized by increased swimming speeds as fish follow or avoid the odorant source ,89. WhenA standardized quantitative analysis of odor-mediated behaviors of zebrafish can be accomplished by recording swimming movements of individual zebrafish before and after odorant exposure in an experimental tank, followed by manual observations or video-tracking software used for behavioral analysis . The latThe increasing number of studies assessing and standardizing behavioral tasks in zebrafish has shown that these responses are robust, consistent, and comparable to other fish species and mammals, as described in the Zebrafish Behavioral Catalog ,86,89. IThe fish olfactory sensory epithelium (OE) is in direct contact with the aquatic environment, and thus it is particularly vulnerable to exposure to waterborne pollutants, chemical toxicants, and direct injury. Damage to the olfactory system can impair olfactory function through various mechanisms affecting its different components: (1) by acting as olfactory signals  ; (2) by Given that odors mediate behaviors essential for survival, the olfactory system is equipped with wide-ranging neuroplasticity, remodeling, and regeneration mechanisms following damage ,18,19,202+ and Na+ channels, either by blocking the current or by modifying the channel-gating properties; these alterations modify neuronal excitability and disrupt intracellular Ca2+ homeostasis, which can lead to cell death [The study of the hazardous effects of heavy metals on olfaction and odorant-mediated behaviors is of particular interest, since these are widespread, neurotoxic airborne and waterborne pollutants resulting from industrial activities, nuclear energy production, mining, military activities, and fertilizer use, among other anthropogenic activities. Metal ions are particularly neurotoxic since they affect the activity of Call death ,111. Morll death ,113.Several groups have investigated the harmful effects of heavy metal exposure to the olfactory system of zebrafish A. CadmiuThese studies clearly show that exposure to waterborne heavy metals produce significant damage of the olfactory organ leading to olfactory dysfunction in zebrafish, with bile salt-sensing ciliated OSNs presenting the most susceptibility to the effects of some of these neurotoxicant agents . The funIn addition to the effects of well-known neurotoxicants such as heavy metals, the olfactory system can be damaged by exposure to numerous chemical compounds and endogenous toxins, as well as by physical injury. Both chemical and physical lesions to the olfactory organs can result in olfactory epithelial damage, which severely impairs olfactory function and modifies odorant-driven behaviors .Pesticides are widespread agricultural and domestic pollutants that have been strongly associated with olfactory impairment in several fish species. Chlorpyrifos (CPF) is a commercially available organophosphate pesticide extensively used for controlling agriculture and household pests. Exposure to CPF downregulates several genes related to olfactory sensing as well as neuronal growth, repair, and regeneration in the olfactory system of zebrafish . RotenonSurfactants are extensively used chemicals found in detergents, emulsifiers, foaming agents, among other commercially available products. Surfactants are routinely discarded on land and water systems and some of them can be toxic and produce olfactory impairment in fish . Both acIt has been reported that patients with chronic kidney disease present severe olfactory dysfunction, although it is not known which uremic toxins produce this effect . StudiesAn experimental paradigm for physical lesioning of the olfactory rosettes is acute and chronic wax plug insertions in the nasal cavity. These interventions produce significant and progressive damage of the olfactory organ, OSN death, and inflammation . MechaniAn important and consistent observation emerging from these studies is that ciliated OSNs, responsive to bile salts, present an increased susceptibility to a diverse variety of toxicants and chemicals. It has been proposed that microvillous OSNs, which respond to amino acids, may present protective mechanisms that render them more resistant to damage, in order to preferentially preserve food sensing and feeding behaviors. It has also been suggested that these neurons may be replaced faster or in greater numbers and, therefore, their numbers do not plunge following olfactory epithelium damage ,22,124.In sum, evidence of the deleterious effects observed in the olfactory organ of zebrafish due to chemical exposure and direct injury supports the use of zebrafish as a robust model for studying the effects of an increased variety of toxicants and direct lesion paradigms in olfactory function.The blood–brain barrier (BBB) is a selective semipermeable barrier composed of the microvasculature of the CNS, endothelial cells, astrocytes, and pericytes, that tightly regulates the passage of molecules from the blood to the cerebrospinal fluid (CSF) surrounding the brain and spinal cord. This barrier is critical for maintaining CNS homeostasis and for protection against toxicants and pathogens present in the blood, inflammation, and disease. However, it is well known that molecules administered to the olfactory epithelium can reach the brain, bypassing the BBB via the olfactory and trigeminal nerves ,145. In It is well documented that olfactory exposure to heavy metals promotes their localization in the olfactory bulb in several fish species. Cd, Ni, and Zn are transported from the OSNs through axonal transport, where they reach the olfactory bulb ,150,151.One of the consequences of OSN death produced by damage to the olfactory organ is the loss of projections of afferent axons from the OSNs to the olfactory bulb, in a process known as deafferentation. Olfactory deafferentation is used as a paradigm to study the influence of olfactory information in the development and maintenance of the olfactory bulb. Since zebrafish offers an amenable model for achieving chemical and physical damage to the olfactory rosettes, it has been widely used to study the effects of the loss of afferent input to the olfactory bulb.Damage to the olfactory organ caused by chronic Triton X-100 exposure produces notable deafferentation of the olfactory bulb, leading to a significant reduction in bulb size and afferent activity ,42. MediDeafferentation of the olfactory bulb by physical injury to the olfactory organ is a common method of examining afferent–target interactions . PhysicaOverall, the results from these studies demonstrate that nasal exposure to heavy metals and toxicants, as well as physical damage to the olfactory organ, can lead to profound alterations in the olfactory bulb via axonal transport or deafferentation mechanisms . These rOne outstanding feature of the zebrafish olfactory system is its extensive ability to regenerate and repair lesions swiftly following injury. Both the olfactory organ and the olfactory bulb present extensive remodeling and repair mechanisms as well as continuous generation of new OSNs and bulbar neurons throughout the organism lifespan ,40,41,42The olfactory rosettes show bimodal patterns of OSN recovery following Cu exposure. On one hand, acute Cu exposure targets ciliated OSNs, with an observed recovery of this cell population after 72 h, whereas chronic exposure produces both ciliated and microvillous OSN loss with a faster proliferation and regeneration of microvillous OSNs observed after 72 h. Olfactory-mediated behavior assessment showed a partial functional recovery of ciliated OSNs, suggesting that although ciliated OSNs are more susceptible to Cu exposure, they are able to recover faster, perhaps through repair mechanisms rather than neurogenesis, a mechanism that was observed for the replenishment of microvillous OSNs .Furthermore, both the olfactory organ and olfactory bulb can rapidly and completely recover to control morphology and activity following different types of damage to the olfactory epithelium: (1) acute Triton X-100 exposure; (2) chronic Triton X-100 exposure; and (3) progressive physical injury with wax plug insertions . FollowiThe remarkable ability of the adult zebrafish olfactory organ to regenerate following different types of damage and lesioning can be explained since the olfactory epithelium presents continuous neurogenesis, due to the presence of neuronal precursors in the basal cell layer of the epithelium. In control conditions, OSNs present a constant turnover rate and most proliferation occurs in discrete clusters located in the apex of the lamellae ,40,152. Furthermore, it was shown that there is an increase in cellular proliferation in the olfactory bulbs and adjacent ventricular zone following an olfactory organ lesion, and that non-neuronal newly born cells likely contribute to the recovery of olfactory bulb volume. On the other hand, most newly born neurons generated following OSN damage do not survive nor integrate into bulbar circuits. It has been shown in mammals that the survival and integration of newly generated neurons in the olfactory bulb requires adequate sensory input during a restricted time window ,154. ThuCumulatively, these studies show that both the olfactory organ and olfactory bulb of zebrafish show extensive regeneration and recovery abilities following peripheral damage with a variety of injury paradigms . MoreoveGiven the widespread occurrence of airborne and waterborne olfactory toxicants such as heavy metals, pesticides, surfactants and general pollutants, there is a pressing need to understand the effects of these harmful compounds on olfactory function. The studies reviewed here shed light on the anatomical, morphological and functional architecture of the zebrafish olfactory system, as well as describing the impact of chemical and physical injury on olfactory function. Furthermore, the regeneration and repair abilities of the zebrafish olfactory system allow for the study of regeneration mechanisms absent or reduced in mammals. Studies reviewed here improve our understanding of diverse fields, including molecular basis of olfaction and olfactory-mediated behavior, olfactory toxicity, neuroplasticity, regeneration mechanisms following damage, among others. These will also provide a foundation for further studies on these topics, with several implications for understanding human health and disease. In conclusion, the cumulative evidence presented here validates the use of zebrafish as a robust model for studying the molecular basis of olfaction, as well as the consequences of toxicant exposure and physical damage to the olfactory organ.


There has been a growing interest in metallic biomaterials during the last five years, as recent developments in additive manufacturing (=3D printing), surface bio-functionalization techniques, infection prevention strategies, biodegradable metallic biomaterials, and composite biomaterials have provided many possibilities to develop biomaterials and medical devices with unprecedented combinations of favorable properties and advanced functionalities. Moreover, development of biomaterials is no longer separated from the other branches of biomedical engineering, particularly tissue biomechanics, musculoskeletal dynamics, and image processing aspects of skeletal radiology. In this editorial, I will discuss all the above-mentioned topics, as they constitute some of the most important trends of research on metallic biomaterials. This editorial will, therefore, serve as a foreword to the papers appearing in a special issue covering the current trends in metallic biomaterials. The research on metallic biomaterials has recently enjoyed a surge in interest, as specific developments, particularly maturation of additive manufacturing (=3D printing) techniques, have provided a host of new possibilities. Due to their high mechanical properties, metallic biomaterials have been used for applications in which load-bearing capability is the major deciding factor. Skeletal applications have, therefore, been the main areas of interest, as the vast majority of orthopedic implants are made from metals, at least partially.Similar to other types of biomaterials, the most important aim in the development of metallic biomaterials is to achieve multiple advanced functionalities. In the case of orthopedic implants, those advanced functionalities should ultimately result in improved longevity of permanent implants, enhanced tissue regeneration performance of bone substitutes, more effective reconstruction of large bony defects, and minimized risk of implant-associated infections. The current research trends on metallic biomaterials are, therefore, focused on achieving the above-mentioned goals, and include 1. Application of advanced additive manufacturing techniques to design and fabricate biomaterials with favorable properties and performance; 2. Bio-functionalized surfaces that could improve bone regeneration performance while minimizing the risk of implant-associated infections; 3. Introducing biodegradation into metallic bone substitutes; and 4. Development of composite biomaterials where metallic biomaterials are combined with ceramic or polymeric materials. Every one of these trends will be discussed in the following sections of this editorial. Another important trend that has emerged during the last few years is application of the techniques developed in the other branches of biomedical engineering, particularly tissue biomechanics, musculoskeletal dynamics, and skeletal radiology, to rationally design metallic biomaterials with better functionalities. We will discuss the relationship between metallic biomaterials and the related areas of biomedical engineering research in the last section of this editorial. As a whole, this editorial aims to present a global picture of some of the most important research trends in the metallic orthopedic biomaterials community. This should provide the context for the papers that follow in this special issue of the International Journal of Molecular Sciences, titled “Current Trends in Metallic Biomaterials: From Additive Manufacturing to Bio-Functionalization, Infection Prevention, and Beyond”.E < 1–2 GPa), to values in the range of those observed for cortical bone  and beyond [Metal 3D printing, otherwise known as metal additive manufacturing, has emerged during the last decade as one of the most successful types of printing techniques for fabrication of functional medical devices. Prior to the advent and maturation of additive manufacturing, the research into metallic biomaterials had become somewhat stagnant, as early developments had made their way into clinical applications, while the possibilities for further developments seemed somewhat limited. Consequently, many research groups were focusing on polymeric and ceramic biomaterials. The tremendous success of metal printing has, however, changed the research landscape since then. In particular, additive manufacturing presents a unique opportunity for adjusting the mechanical properties of metallic biomaterials using topologically ordered volume-porous biomaterials. The mechanical properties of the metallic alloys used for fabrication of orthopedic implants are usually much higher than those of the native bone tissue, increasing the risk of stress shielding ,2,3,4. Td beyond ,7,8.In addition to adjusting the mechanical properties, topologically ordered volume-porous metallic biomaterials could be used for adjusting the other types of properties that are important for their application as orthopedic implants and bone substitutes. That includes fluid flow and mass transport properties  ,10,11 anMoreover, additive manufacturing techniques have rendered fully patient-specific implants as a feasible design option chemical or physical mechanisms. The techniques based on chemical and biochemical agents have been most extensively researched. In particular, it has been shown that the local delivery of specific growth factors, such as bone morphogenic protein (BMP), could result in improved tissue regeneration performance ,28,29. MOn the physical side, recent discoveries have shown that stem cell fate could be determined with nanoscale topographies present on the surface of biomaterials ,32,33. TUltimately, it may be important to combine the (bio)chemical approach with the physical one to maximize the tissue regeneration performance of metallic biomaterials while minimizing the risk of drug-related toxicities. This line of research has not received much attention, as of yet, and could be a worthy research avenue for future studies.Several studies appearing in this special issue address bio-functionalization, biocompatibility, and bioactivity of  biomaterials ,40,41,42Prevention and treatment of implant-associated and biomaterial-associated infections are one of the priorities in orthopedic surgeries. This is particularly important for the most vulnerable patient groups, such as orthopedic oncologic patients whose immune system may have been compromised due to chemotherapy and/or radiotherapy, or trauma patients who have to undergo a long surgery with a large open wound that is needed for implantation of large implants. In many such cases, metallic biomaterials that minimize the risk of infections could potentially play an important role.Similar to the case of bio-functionalization techniques aimed towards improvement of bone tissue regeneration performance, the biomaterials that minimize the risk of infection may work on the basis of (bio)chemical antibacterial agents or physical mechanisms. The (bio)chemical approaches are usually based on antibiotics , antimicOn the physical side, nanoscale patterns, including those with large aspect ratios, are found to not only affect bacterial adhesion to the surfaces, but also kill bacteria . MoreoveAlthough biodegradable metallic biomaterials have been under development for quite some time ,62,63,64Similar to the case of mechanical properties, the biodegradation profile, as well as the characteristics of the biodegradation products, could potentially be adjusted using the topological design at the microscale. In addition, the large surface area of topologically ordered volume-porous biomaterials could be used to speed up the biodegradation process, thereby facilitating the application of metals that degrade too slowly, e.g., zinc or iron. The relationship between the microscale topological design and biodegradation profile, however, remains largely elusive.Design of new (meta-)biomaterials with novel properties requires application of techniques developed in the other branches of biomedical engineering and skeletal radiology. Two particular types of expertise are notable in this regard, namely, biomechanical modeling and medical image processing.The biomechanical models used in orthopedic biomechanics could be divided into two main subcategories: 1. Tissue biomechanics and 2. Musculoskeletal dynamics. Tissue biomechanics modeling allows for characterization and study of the mechanical and physical  behaviors of biological tissues. Since mimicking the properties of the native tissue is often considered the ultimate goal of the biomaterials that are used to replace them either temporarily or permanently, this type of characterization is crucial to the design of new biomaterials. In the vast majority of cases, tissue biomechanics models ,69,70,71Image processing techniques are required to obtain the shapes of the bones and other skeletal tissues at different scales. Moreover, tissues with different  densities attenuate the X-ray beams differently, thereby allowing use of the gray scale data of the resulting images as a way to estimate the distribution of the tissue density. Combining the data regarding the shape and density distribution of bones is crucial for building computational tissue biomechanics models discussed above. Moreover, advanced image processing techniques, such as those based on statistical shape models and statistical shape and appearance models  could faIdeally, one would like to combine image processing techniques with musculoskeletal dynamics models and tissue biomechanics models, to develop a streamlined approach for design of meta-biomaterials based on quantitative data regarding the shape, density distribution, loading conditions, micro-architectural design, and mechanical properties of tissues and organs. The same type of data could be used for designing patient-specific implants and evaluating the designed implants to see whether the resulting stress/strain distributions are comparable with those observed in the native tissues. This integrated approach is a relatively new line of research, particularly in connection with the design of biomaterials, and could result in some new ways of designing biomaterials and implants that are based on physical models and quantitative data, as opposed to the heuristic approaches used in the past. One of the review papers appearing in this special issue addresses some of the above-mentioned topics .Even though metallic biomaterials offer a host of favorable properties whose ranges could be further expanded using rational design techniques and additive manufacturing, other categories of biomaterials, including ceramics and polymers, could add additional functionalities or broaden the possible ranges of properties. That is why composite biomaterials combining different categories of biomaterials could be of great interest and value. The research into this category of biomaterials has been ongoing for several years . HoweveWe briefly reviewed some of the current trends of research on metallic biomaterials, particularly the metallic biomaterials aimed for skeletal applications, such as permanent orthopedic implants or the temporary bone substitutes used for treatment of (large) bony defects. The topics identified here are currently being intensively researched, and could lead to breakthroughs in treatment of skeletal diseases. The relevant literature, including the papers published in this special issue as well as those published elsewhere, were cited. Following up on the research areas suggested here, with new ideas and techniques, could further stimulate the success of metallic biomaterials community as a whole, while offering new generations of metallic medical devices.


They provide a deeper understanding of the mechanisms involved in metal and metalloid toxicity and highlight the need to develop strategies to decrease exposure to metals, as well as identify substances that may help overcome the hazardous effects within the living body.A general disregard by industries and big corporations for the environment has meant that the natural environment has repeatedly been polluted, jeopardizing the well-being of all living organisms. The toxic effects of metals and metalloids have been recognized for quite some time to generate several free radicals and other reactive species that lead to cellular oxidative stress. Metals and metalloids are toxic elements at the top of the priority list of hazardous substances of the Agency for Toxic Substances and Disease Registry (ATSDR). Gaps of knowledge still exist that are related to their toxicity, mainly concerning the mechanisms of action. Due to their extreme potential to cause oxidative damage, the structural, functional, biochemical, and molecular effects of metals and metalloids on oxidative stress need description, and the identification of new potential competitive-protective agents is imperative. This special issue brings together the results of six papers covering several aspects of the toxicology of metals and metalloids in Rosmarinus officinalis), was explored to rescue the arsenite-induced oxidative stress in hepatic cells in in vitro and in vivo models in the study of S. Das et al. Carnosic acid was able to significantly rescue the activation of MAPK, NF-κB, p53, and intrinsic and extrinsic apoptotic signaling seen in arsenite-exposed hepatic cells. Carnosic acid could also significantly counteract with redox stress and ROS-mediated signaling, thereby attenuating arsenite-mediated hepatotoxicity. Other effects, such as arsenic bioaccumulation, DNA fragmentation, and hematological changes, were also reverted in models after coadministration with carnosic acid. The authors attributed the rescue to the antioxidant potential of carnosic acid which in their view had the potential to be a new therapeutic agent to counteract with arsenite-mediated toxic manifestations. In the same way, the study of A. Officioso et al. compared the beneficial effects of olive oil phenol hydroxytyrosol (HT) and 5-S-lipoylhydroxytyrosol (Lipo-HT) on oxidative alterations of human erythrocytes induced by exposure to inorganic mercury. Both HT and Lipo-HT decreased Hg-induced generation of ROS, hemolysis, and the depletion of intracellular GSH levels. At all doses tested, Lipo-HT exhibited a higher ability to counteract Hg-induced cytotoxicity compared to HT. To explain that, the authors propose a model where Lipo-HT is more effective to chelate mercury ions and encourage the use of Lipo-HT in nutraceutical strategies to counteract heavy metal toxicity in humans.Carnosic acid, a natural benzenediol abietane diterpene found in rosemary (MutL homolog (MLH1) and the DNA methyltransferase 1 (DNMT1) was noticed, accompanied by an increase in global methylation by LINE-1. The authors suggest that vitamin E supplementation has a high potential for treatment and might even be used as a possible approach in the prevention of diseases caused by obesity and diabetes.Obesity- or diabetes-induced oxidative stress is discussed as a major risk factor for DNA damage. What has not been addressed yet is how vitamin E and many polyphenols exhibit antioxidative activities with consequences on epigenetic regulation of inflammation and DNA repair. To shed light on that, the study of K. Zappe et al. investigated the counteraction of oxidative stress by vitamin E in the colorectal cancer cell line Caco-2 under normal- and high-glucose cell culture condition. Results revealed a dose-dependent counteracting effect of vitamin E on malondialdehyde levels. Also, an induction in the expression of the genes of DNA repair Cadmium is one of the most poisonous environmental chemicals, causing toxicity in humans and experimental animals. The study of M. C. Cupertino et al. investigated the relationship between germ and Leydig cell death, testosterone, and adiponectin levels in cadmium-mediated acute toxicity. Dose-dependent cadmium accumulation in testes was identified. At the highest doses of exposure, animals exhibited marked inflammatory infiltrate and disorganization of the seminiferous epithelium. Although Leydig cells were morphologically resistant to Cd toxicity, massive germ cell death, DNA oxidation, and fragmentation were observed. Also, testosterone and adiponectin levels were significantly decreased by cadmium. The authors discuss the mechanisms behind the findings, but why germ cells are a primary target of cadmium-induced toxicity while Leydig cells remain resistant to death even when exposed to higher doses of this metal is still unclear.2 layer or using new, biodegradable, and more biocompatible materials. The authors also suggest that supplementation with antioxidants could be tested as a therapeutic procedure in patients treated with titanium fixations.Despite the undoubted advantages of titanium bone fixations such as high biotolerance, favorable mechanical properties, and osseointegration ability, their negative impact on the human body, both at the implant site and at the systemic level, is still debated. To contribute to the toxicological background in this emergent area, the study of J. Borys et al. evaluated the influence of Ti6Al4V titanium alloy on redox balance and oxidative damage in the periosteum surrounding the titanium miniplates and screws as well as in plasma and erythrocytes of patients with mandibular fractures. The occurrence of a redox imbalance as well as oxidative damage in the periosteum surrounding the Ti6Al4V titanium alloy was demonstrated. Changes in antioxidant potential in the patients treated with titanium implants were also observed in the plasma/erythrocytes. Results indicate the need to improve the miniplates and screws used for osteosynthesis by increasing the thickness of the passive TiOFinally, S. R. Lee critically reviewed the role of zinc as either an antioxidant or a prooxidant. For instance, it functions as an antioxidant through the catalytic action of copper/zinc-superoxide dismutase, stabilization of membrane structure, protection of the protein sulfhydryl groups, and upregulation of the expression of metallothionein, which possesses a metal-binding capacity and exhibits antioxidant functions. Also, zinc can suppress anti-inflammatory responses that would otherwise augment oxidative stress. On the other hand, zinc deficiency and zinc excess cause cellular oxidative stress. The conditions behind the dual actions of zinc and the oxidative imbalance that occurs in zinc deficiency and zinc overload in conjunction with the intracellular regulation of free zinc are summarized in this review.We hope that the new findings on the toxicity of metals and metalloids as well as on the identification of protective agents presented in this special edition can contribute to the development of science, to the environmental and health regulatory agency policies, and to more effective strategies to rescue toxicity.


This case report describes the orthodontic treatment used to prepare for restorative dentistry, aimed at the functional rehabilitation of an adult patient with Class I malocclusion, right posterior crossbite, and significant tooth wear on the palatal face of the maxillary canine to canine. The orthodontic treatment was performed with a total fixed appliance mini-expandex-type expander, associated with vertical elastics. Good dental intercuspation was obtained and enabled conditions for the restoration of the abrasions on the palatal surfaces of the maxillary incisors and canines and the tips of the buccal cusps of the maxillary right premolars and first molar. Excellent results were found during a 37-month follow-up. Gradual attrition of the occlusal surfaces of the teeth appears to be a general physiological phenomenon in all mammals, in every civilization, and at all ages . Tooth wJang et al. reported that quantitative analysis of tooth wear is a function of orthodontic treatment modality . Other sThe aim of this article is to describe the orthodontic treatment, and functional rehabilitation, of an adult patient, skeletal and dental Class I, with posterior crossbite and significant abrasion on the palatine face of the maxillary canines, incisors, and right premolars and molars.A 38-year-old man came for orthodontic treatment with tooth abrasion as his chief complaint. His medical and dental histories showed good general health, except for a stressful lifestyle. He did not report any mandibular movements with nocturnal bruxism but did report functional movements or clenching in the daytime. He also stated that, when brushing, he applies too much force.Dental analysis showed an Angle Class I malocclusion, normal overjet, a moderate overbite, and a slightly maxillary midline, shifted to the right (a posterior crossbite on the right side involved the first molar and both bicuspids . GingivaZ-angle = 78°) , brachyfacial pattern (FMA = 17°), upright mandibular incisors (1-NB = 15°), and a concave profile (e = 78°) . The pane = 78°) .The primary goals of the treatment proposed were as follows: transverse skeletal expansion of the maxilla, creation of a condition to restore the palatal side of the maxillary canine and incisors, and transverse dentoalveolar expansion of the maxillary right side.Based on the treatment objectives, the following treatment options were suggested: surgically assisted maxillary expansion, associated with a hyrax-type expander; transpalatal arch; intermaxillary elastics; expansion with arches and intermaxillary elastics; or mini-expandex-type expander for dentoalveolar expansion.The patient did not accept the surgically assisted palatal expansion, so the first step was to affix the fixed mini-expandex-type expander to the maxillary first molars. On the left side, an extension was established on the second premolar and the second molar to increase the anchorage Figures . The actAfter completing the orthodontic treatment, three sessions of dental whitening were performed, with 38% hydrogen peroxide. The technique of direct restoration of the composite resin in the anterior  and posterior  teeth, using a universal adhesive system, allowed a better control of the restorative sequence . The resThe posttreatment records showed the correction of the maxillary transverse deficiency and the restoration of palatal surface abrasion of the maxillary canine and incisors. Ideal overjet and overbite relationships were maintained, with good intercuspation. The buccal cuspids of the maxillary right molar and premolars were built-up with composite resins . RetentiThe cephalometric analysis did not show changes in the skeletal relationships and did show maintenance of the incisors' inclinations Figures . The panAfter 37 months, follow-up photographs showed a stable occlusion, with maintenance of the overjet and overbite, as well as the molar and canine relationships, except for a slight rotation of the mandibular left central incisor. The patient stopped using the retainer one year after debonding .This clinical report demonstrates an approach to restoring severely worn maxillary anterior teeth with minimum dental preparation and adhesively bonded facial and palatal composite resins.Tooth wear on malocclusion subjects should not be considered pathological but rather the consequence of different interocclusal arrangements . In thisSuccessful dentoalveolar maxillary expansion with a mini-expandex-type palatal expander, associated with vertical elastics, was achieved, enabling restoration of the abraded teeth. The restorative management of the worn dentition involved working with a conformative treatment, involving restoration of the affected teeth without changing the occlusal vertical dimension and conforming to the intercuspal occlusal relationship that the patient presented .Tooth wear can be described as the loss of hard dental tissue, resulting from physical or chemical attack; it is an all-embracing term, used to describe the combined processes of abrasion, erosion, and attrition .Our patient presented with a strong brachyfacial pattern, and the tooth wear was evident on the crossbite side and into the palatal surface of the maxillary canines and incisors. This differs from the results of Kiliaridis et al., who found no significant differences in the sagittal relationships between persons with advanced wear and normal standards .A comprehensive treatment plan was established, with coherent objectives and a clear understanding of the patient's expectations, including an assessment of the patient's ability to adapt to the treatment at the beginning, and involving a multidisciplinary approach to achieve and maintain a successful outcome.After 37 months, follow-up photographs showed a stable occlusion, with maintenance of the overjet, overbite, restorations, and the molar and canine relationships


This review is devoted to the medical application of tetrodotoxin (TTX), a potent non-protein specific blocker of voltage-gated sodium (NaV) channels. The selectivity of action, lack of affinity with the heart muscle NaV channels, and the inability to penetrate the blood–brain barrier make this toxin an attractive candidate for anesthetic and analgesic drug design. The efficacy of TTX was shown in neuropathic, acute and inflammatory pain models. The main emphasis of the review is on studies focused on the improvement of TTX efficacy and safety in conjunction with additional substances and drug delivery systems. A significant improvement in the effectiveness of the toxin was demonstrated when used in tandem with vasoconstrictors, local anesthetics and chemical permeation enhancers, with the best results obtained with the encapsulation of TTX in microparticles and liposomes conjugated to gold nanorods. The medical community defines neurotoxins as substances that damage, destroy or disrupt the function of nervous tissue at low concentrations. The very concept of ’neurotoxins’ does not assume that they have a purpose, although these substances provide humankind with essential drugs, such as analgesics, anesthetics, and muscle relaxants, which are crucial for modern medicine, especially within the surgical domain. The acute and instantaneous effect on the nervous system at concentrations far lower than their median lethal dose (LD50) allows minimal concentrations of neurotoxins to be used to reach the desired effect . HoweverChondrodendron of the Menispermaceae family and the genus Strychnos of the Loganiaceae family, and are muscle relaxants that competitively bind acetylcholine receptors [Clostridium genus on the extracellular binding of glycoproteins on cholinergic nerve endings and the intracellular blockade of acetylcholine secretion, is well-founded [Papaver somniferum poppy, was a revolutionary discovery in the field of pain management. A wide spectrum of toxin action, in both the peripheral and central nervous systems, is due to the ability to activate μ-opioid receptors on neuronal cell membranes [Neurotoxins do not just have a long history of application in medical practice, but have also initiated new directions in drug design and development. In this context, it is worth noting curare, botulinum toxin, and morphine. Curare is a collective name for a group of toxic alkaloids, isolated from plants of the genus eceptors . The moseceptors . Perhaps-founded . The use-founded . Botulin-founded . Anotherembranes . In spitConus. A wide range of analgesics for treating various types of pain are being developed on the basis of conotoxin peptides, including ziconotide, χ-conotoxin MrIA and leconotide [Various neurotoxins, at different stages of research, development and introduction into therapeutic practice, are also of a great interest. For example, a bacterial tetanus toxin possesses a unique ability to penetrate the central nervous system through motor neurons, making it a putative neurotropic agent that could facilitate the transport of therapeutic compounds . Other sconotide . The neuconotide  and Austconotide , are alsModern research in the field of neurotoxin drug development focuses on specific blockers of voltage-gated sodium (NaV) channels, such as µ-conotoxin , saxitoxThis review is devoted to the medical applications of TTX, with a special emphasis on studies focused on the improvement of its efficacy and safety in conjunction with additional substances and drug delivery systems.In recent decades, the concept of pain has broadened and is currently attributed to a wide variety of problems. People suffering from pain may experience acute, chronic, or intermittent pain, usually caused by surgical intervention, trauma, cancer, osteo- and rheumatoid arthritis, spinal problems, neurodegenerative diseases, and other causes . HoweverUntil the 1960s, opioids  were the main analgesic drugs in medical practice. The analgesic effect of such drugs is caused by their ability to inhibit the release of neurotransmitters from the primary afferent fibers in the spinal cord and to activate the descending nociceptive inhibitory control in the midbrain  Figure . Despite+-selective ion conduction, voltage-dependent activation and inactivation, and specific binding sites for different natural toxins [NaV channels, large integral membrane proteins, play a key role in pain signal transmission from nociceptors to the central nervous system, being responsible for the initiation and propagation of action potentials in excitable cells by allowing the influx of sodium ions . The α-sl toxins . α-subunl toxins .Specific blockers of NaV channels are of great interest in the development of new analgesic drugs. Normally, nociceptors possess five types of sodium channels  neurons), expressed in various injuries of the peripheral and central nervous systems ,23. The Among all NaV channel blocking substances studied for medical purposes, the most effective is TTX, which selectively blocks six out of nine known NaV channels, including NaV1.3 and 1.7, mentioned above  Figure . The feaDiscovered in 1909 in the ovaries of globefish (family Tetraodontidae), TTX was subsequently isolated from a variety of marine and some terrestrial animals, marine algae, and bacteria ,38,39. TNumerous studies have been conducted that address the efficacy of TTX against pain in a number of experiments using animal pain models and in clinical trials in humans . The maiA number of studies have shown the effectiveness of the toxin in preventing hyperalgesia, mechanical allodynia, and the spontaneous development of afferent activity, caused by injured sciatic and infraorbital nerves in rats ,53 and mTTX’s effectiveness against visceral pain was also studied ,65,66. AThe main obstacle to the introduction of TTX into clinical practice is the potential for the occurrence of toxicity due to the systemic distribution of the drug, which, in the worst case, can lead to diaphragmatic nerve blocking and result in respiratory tract paralysis . Most ofThe efficacy of NaV channels in blocking toxins, including TTX, is limited due to their relatively weak penetration to the site of action through different tissue barriers . High coA number of studies were conducted to improve the TTX therapeutic index using adjuvants that increase the penetration of small molecules through biological barriers. A group of compounds, so-called chemical permeation enhancers (CPE), that include a big class of surfactants and a wide range of chemical reagents that are not surfactants , is capable of reversibly modifying lipids, leading to an increase in membrane fluidity . A comprIn addition to the CPE vasoconstrictors, epinephrine is used to increase the efficacy of TTX ,75,76. RTypical local anesthetics, widely used in medical practice, are another group of substances that can prolong the effects caused by TTX. Adams et al. showed that the simultaneous administration of TTX with various local anesthetics  increased the sciatic nerve block rate to 100% and prolonged its average duration by 2.5–5 times in comparison with the test anesthetics alone . SeveralAnother approach is the use of delayed-release drug systems that significantly prolong the period of the toxin exposure to the nerve and allow its high local concentration to be maintained ,83.One of the most common methods of targeted delivery is local anesthetic encapsulation in microparticles consisting of biodegradable polymeric particles, for example, poly (lactic-co-glycolic) acid (PLGA) . Kohane 2 for 10 min, once a day for four days. At the same time, the duration of the local anesthesia decreased with each subsequent irradiation cycle, indicating a decrease in the amount of the drug released over time. The disadvantage of this technique is a significant decrease in the effectiveness of the excitation of liposomes under the influence of light caused by the deepening penetration and the increased level of light flux power, which can lead to tissue damage [2 and avoiding phototoxicity.Another research area is remotely-triggered drug delivery systems, in which the administered drug is re-activated by a secure external trigger, such as an electromagnetic field, ultrasound or near-infrared light (NIR) ,86. Zhane damage . In anotDifferent alternatives for the improvement of TTX efficiency are illustrated in ®, aimed at chronic pain and neuropathy caused by cancer or chemotherapy treatment, completed phase II and III clinical trials in the USA and Canada [Discovered in 1909, TTX still attracts researchers’ attention due to its high activity and ability to selectively block the NaV channels involved in pain signal formation. The selectivity of action and the inability to penetrate the central nervous system provide TTX with significant advantages over other analgesic and anesthetic drugs. TTX is a part of a number of experimental drugs developed by Wex Pharmaceuticals. One such drug, Tectind Canada .The introduction of TTX into the pharmaceutical market largely depends on the further success of its drug formula development. Numerous studies reviewed in this article demonstrated a significant improvement in the effectiveness of TTX when used in tandem with other agents, in particular, CPE, vasoconstrictors, and local anesthetics . The foc


Botulism outbreak due to consumption of food contaminated with botulinum neurotoxins (BoNTs) is a public health emergency. The threat of bioterrorism through deliberate distribution in food sources and/or aerosolization of BoNTs raises global public health and security concerns due to the potential for high mortality and morbidity. Rapid and reliable detection methods are necessary to support clinical diagnosis and surveillance for identifying the source of contamination, performing epidemiological analysis of the outbreak, preventing and responding to botulism outbreaks. This review considers the applicability of various BoNT detection methods and examines their fitness-for-purpose in safeguarding the public health and security goals. Clostridium botulinum, C. argentinense, C. butyricum and C. baratii spp). Among the seven known BoNTs serotypes (serotype A–G), type A, B, E, and F are commonly linked to cause food-borne botulism in humans, while type C and D are commonly associated with botulism in animals and birds. BoNTS are the most poisonous substances known  proteins, crucial for the synaptic vesicle membrane fusion and neurotransmitter release during neuroexocytosis. The tridomain structural organization and the trimodular function of BoNTs contribute to three major steps of BoNT intoxication process: receptor-mediated endocytosis, membrane translocation across the endosomes, and inhibition of acetylcholine release by cleaving SNARE proteins  are metalloendoproteases produced as protein complexes by anaerobic spore forming neurotoxigenic clostridia  wound-borne botulism, in which spores grow to produce and release of toxin within injured tissues  exhibit dryness of mouth, double vision, blurred vision, drooping of upper eyelids, difficulty in swallowing and speaking, dysarthria and generalized weakness. This cluster of signs and symptoms allows physicians to distinguish cases of botulism from more common conditions often seen with other foodborne illnesses. Patients with severe cases of botulism exhibit bilateral flaccid paralysis of arms and legs and difficulty in breathing, due to paralysis of the respiratory muscles  and/or suspected food sources  is routinely used standard method to detect and confirm the presence of BoNTs in clinical specimen and food or environmental samples linked to botulism outbreaks. One of the benefits of the MLB is also that it holistically represents the trimodular domain function, and demonstrates the biological activity of BoNTs to cause botulism. This includes simulating the symptomatic progression of (untreated) human botulism, as they lead to paralysis and death, using mice  alerting the public about the food sources linked to outbreaks; (2) notifying and monitoring producers; (3) enforcing food safety regulations; (4) identifying the means by which food has become contaminated, whether on the farm, during processing, shipment, storage, or preparation to eliminate those sources of contamination; (5) enhancing regulatory tools with validated new technologies; and (6) refining the guidance for good agricultural and/or manufacturing practices (USFDA)The goal of source tracing investigations in response to an outbreak is to identify which food sources have become contaminated by biological hazards to eliminate those foods from the distribution system or commerce, as quickly as possible. Source tracking involves linking clinical cases or clusters of illnesses, often reported from different geographic areas to a common source. Detecting the presence of toxin in food samples is a reliable approach for source-tracking, since it is the pre-formed toxin that causes an outbreak. Regulatory decisions such as recalls, seizure, injunction, administrative detention of (food) products, civil money penalties and/or prosecution and license suspensions or declarations of public health emergencies must be legitimately grounded on robust laboratory methods that can reliably confirm the presence of a definite health hazard that harm public health. Just as it is important not to underestimate the risk to public health posed by food-borne botulism outbreaks, it is also important not to cause unnecessary panic, food waste, or economic losses to the food industry or food commerce  are needed to stimulate the growth of clostridia and toxin production on food substrates , or pretreatments meant to reduce pathogen populations, and the presence of competing organisms , are known to inhibit the growth of C. botulinum grow. Our present knowledge about the in vitro stability and potency of BoNTs, which are known to be influenced by the composition of the toxin complex containing non-toxic, accessory proteins , and limit the ability the toxin to cause botulism outbreaks. Our understanding of the toxin (complex) formation, its stability and potency in food or environmental sources is still limited. BoNT stability and potency when produced or released during autolysis on different food substrates can vary and may depend upon the growth phase, strain that produces the toxin. They may also vary or be altered depending upon the physicochemical properties of food sources in which Weissella oryzae . Deliberate release of chemical and biological agents such as sarin gas, botulinum toxin  of BoNT/B in a variety of foods, using type B-specific monoclonal antibodies for immuno-affinity capture  by measuring their enzymatic activity in an immunoassay  and environmental samples; and compare the phylogenetic relatedness of those outbreak strains to support epidemiological investigations. Availability of BoNT gene sequence information allows for the design of sequence specific DNA primers to detect and identify toxin serotypes/subtypes by PCR methods  has demonstrated the high genomic diversity of toxigenic clostridia, leading to the identification of novel genotypes  in pre-processed or post-processed food, or environmental samples to inform risk assessments; support development and implementation of appropriate emergency response plans, and public safety actions . However, sufficiently large networks of outbreak data, high quality reference databases and internationally acceptable quality standards of data and metadata will be critical to build such capabilities for the continuing enhancement of food safety and public health (Sjodin et al., In vitro detection methods like ELISA, electrochemiluminescence assay, enhanced chemiluminescence based immunoassay formats, and endopeptidase activity based Endopep-MS and Endopep-ELISA methods are rapid and highly sensitive methods that can reliably detect toxin antigens and enzymatic activity of the BoNTs in complex sample matrices. Beyond demonstrating the presence of C. botulinum in clinical specimen, these in vitro methods can also provide stand-alone evidence to confirm botulism in suspected patients and are potential alternatives to reduce reliance on the MLB. The statistical gap between the number of laboratory confirmed cases and the clinically diagnosed cases that were reported over several years of time, may possibly and partly originate because of the dependence on activity based assay like MLB, although it is highly sensitive for detecting BoNTs. Tests that only measure activity of the toxin can mislead by giving negative results, if toxin activity diminish due to intervention from physiological or native immune factors, over time. However, it remains to be seen if sensitive immunoassays that can detect toxin-antigens can substantially reduce the occurrence of false negatives or inconclusive results during clinical investigations, when compared to the assays that rely on toxin activity. In vitro toxin detection methods are also highly valuable for screening or presumptive testing of suspected food, environmental samples, or other sample types. Detection of toxin antigens, toxin complex-associated accessory proteins or the enzymatic activity of BoNTs are good indicators for the likely presence of biologically active forms of BoNTs in tested samples. Immunoassay-based BoNT detection methods can also be adapted in to multiplex screening platforms for detecting various other biothreat toxins and pathogens and can be performed in a rapid, high-throughput screening format, amenable for automation.in vitro assays, and next-generation sequencing platforms need to be evaluated for routine use and intended benefit. Fitness of various BoNT detection platforms in terms of standard performance characteristics needs to be validated in multiple laboratories in the framework of their ability to support public health programs, policies and procedures to assure optimal, integrated and informed decision-making processes to achieve the goals of public health, food safety and national security.Rapid changes in food production, processing, and distribution methods, including globalization of the food supply and rising public demand for foods that are “less processed” or “preservative free” contribute to scenarios in which outbreaks of foodborne illness, including botulism, could become much larger and cross international borders. Implementation of prevention-based food safety controls can help prevent major botulism outbreaks. Hence, besides exploring strategies to restrict clostridial growth in foods, better understanding of foodborne-physiological stimuli that directly induce or regulate toxin gene expression at the transcriptional level (Connan and Popoff, NT, EJ, SP, DH, LS, and SS contributed to conception of the review article. NT wrote the first draft of the manuscript. NT, SP, DH, LS, and SS wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.KV is the CEO of Omni Array Biotechnology, Rockville, MD, USA. BS is also the president of Prime Bio, Inc., N. Dartmouth, MA, USA. The remaining 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.


This review summarizes the information on in ovo and post-hatch-feeding of different nutrients and feeds additives and their effects on gut development, histomorphology, microbiology, and immunology. Furthermore, this review will provide insight on the future of early nutrition programming as a strategy to enhance gut health, thereby improving overall health and production so that the poultry industry can benefit from this technique.Healthy gastrointestinal tract (GIT) is crucial for optimum performance, better feed efficiency, and overall health of poultry. In the past, antibiotic growth promoters (AGP) were commonly used to modulate the gut health of animals. However, considering the public health concern, the use of AGP in animal feeding is banned or regulated in several jurisdictions around the world. This necessitates the need for alternative nutritional strategies to produce healthy poultry. For that, several alternatives to AGP have been attempted with some success. However, effective modulation of the gut health parameters depends on the methods and timing of the compound being available to host animals. Routinely, the alternatives to AGP and other nutrients are provided in feed or water to poultry. However, the GIT of the newly hatched poultry is functionally immature, despite going through significant morphological, cellular, and molecular changes toward the end of incubation. Thus, early growth and development of GIT are of critical importance to enhance nutrients utilization and optimize the growth of poultry. Early nutrition programming using both  Poultry production has increased at a faster rate than any other livestock animal globally. Among others, the nutritionally balanced-feeding program along with antibiotic growth promoters (AGP) in poultry diets played a significant role in achieving this success. However, the poultry industry is under pressure to redefine its nutrition program to grow safe and quality meat in the light of public health concern due to the use of AGP in poultry diets. Maintenance or improvement of gut health is essential for optimum growth, better feed efficiency, and overall health of poultry . Also, aGut health covers the efficient nutrient utilization, macro- and micro-structural integrity of the gut, the stability of the microbiota, and the status of the immune system , 3. Morein ovo feeding to specially designed post-hatch diets (in ovo injection of sulfur-containing amino acids (methionine plus cysteine) in the embryonated eggs exposed to heat stress have positive effects on gene expression and antioxidant indices as well as reduce the lipid profile of newly hatched broiler chicks  and the immune system of poultry. Unlike mammals that can influence the development of fetus even after parturition, avian species can leverage expression only through the composition of the egg. Due to this restriction, all necessary nutrients, growth factors, and the machinery needed for the development are required to be present in the fertilized egg. Also, because of a rapidly growing embryo and the fast-metabolic turnover of modern chicks, some of the essential nutrients can become depleted or insufficient during adverse environment and disease challenges. This constraint of nutrient reserves may limit the maximal development and growth of newly hatched chicks. The availability of essential nutrients can be improved, and the existing challenges can be overcome to some extent by providing early nutrition to the embryos and chicks. Early nutrition or feeding in poultry production is a concept of providing the required nutrients to the birds either during the period when the embryo is developing or immediately after hatch until they attain a fully matured digestive system .in ovo feeding (IOF). This technique provides the opportunity to supply essential nutrients, neutraceuticals, and functional foods to uplift the status of growth and development of the embryo. Several routes including amniotic  concept for all the essential amino acids should be applied. The emphasis is not on minimum crude protein but rather on the proper balance or ratio of each amino acid to lysine. The use of synthetic amino acids will be beneficial and can help to prevent excess crude protein. An excess of indigestible protein in the hindgut can predispose to several intestinal challenges .Clostridium perfringens. Toxins produced by Cl. perfringens are responsible for necrotic enteritis . Different cereal grains  have different starch characteristics and physico-chemical properties. For example, wheat contains high levels of non-starch polysaccharides (NSP) such as β-glucans and arabinoxylans . The phynteritis . Therefonteritis –61Salmonella typhimurium and Cl. perfringens in the GIT of the birds  plays a significant role in the avian immune system . Hence, In ovo manipulation of the embryo is not only a method of filling up the reserves with nutrients but it is also a tool that allows injecting other substances that can stimulate the immune system, modify the gut microbiome, and shift the level of production of metabolites. Dibner et al.  increased cell-mediated immunity while the injection of ω-3 fatty acids of marine origin induced a humoral response. Likewise, glucose triggered humoral immunity while fructose and ribose modulated cellular immunity in broilers receiving in ovo injection in yolk sac/amnion on d 14 of incubation (in ovo feeding of amino acids can enhance the growth-related genes and modulate the expression of immune genes in broilers (in ovo (In ovo feeding of arginine to late-term embryo has been documented to increase secretory immunoglobulin A (sIgA) in the intestinal mucosa and activate Arg-NO signaling pathway in broilers (in ovo feeding of arginine can enhance the immunological barrier function of intestinal mucosa and improve the overall immunity and intestinal health of birds.r et al.  proposedouraging , 101. Sim et al.  injectedm et al.  reported Cherian  using incubation . It has broilers . Early i (in ovo . Also, e (in ovo , producebroilers . The incSalmonella enteritica in neonatal chickens (The application of some additives as a nutritional approach to tackle deficiencies during the early feeding period also have some prospect in reinforcing the immunity of growing poults. Feeding of β-glucan can strengthen the innate immunity by up-regulating the oxidative burst, phagocytosis and bactericidal killing capacity of heterophils and decrease the incidence of organ invasion by chickens . Early fchickens , 112. Hochickens . Hence, in ovo technology have established a new scope for perinatal nutrition, allowing and creating new challenges and opportunities for nutritionists to optimize poultry production. The in ovo injection of important nutrients or substances into the amnion is a novel way to feed critical dietary components to embryos. Indeed, in ovo feeding may “jump-start” development, improving the nutritional status of the perinatal chick. The in ovo feeding technique has several advantages, including improvements of total digestive tract capacity; increased body weight, growth rate, and feed efficiency; reduction of post-hatch mortality and morbidity; improvements in the immune system and the response to enteric antigens; reduction in incidence of developmental skeletal disorders; and increase in muscle development and breast meat yield. The next step in the early nutrition could be to imprint genes of a bird at a very early age and turn it into a more efficient animal later. In addition, the administration of digestible nutrients into the amnion of embryos can bring an improvement in bird quality, increased glycogen reserves, fast development of the total digestive tract superior skeletal health, better muscle growth rate, higher body weight gain, improved feed conversion, and enhanced immune function (in ovo feeding.The continuous development and improvement of function . Using nThe main limitations still are associated with embryo development and nutrient metabolism. Another question is a limitation in the probiotic preparation that fits the specific needs of the individual bird. Future early nutrition would be feeding complex symbiotic that would replace feed additives and supplements in the post-hatch feed and is more beneficial to the overall poultry industry.in ovo feeding technique for application at commercial scale in farm condition, understand the embryonic development and nutrient metabolism process more precisely, and understand how early nutrition affects specific genes responsible for performance, intestinal health, and overall health-related traits in poultry.With the increase in productivity and highly feed efficient birds, the nutritional demand of embryos and early aged chicks has changed over decades. Early nutrition programing is one of the latest and successful methods to feed embryos and recently hatched chicks to prepare chickens with the healthy gut, favorable microbiota, improved immunity, and overall improved growth performance. Currently used materials to feed as early nutrition includes probiotics, prebiotics, exogenous enzymes, amino acids, hormones, vaccines, and drugs. Early feeding to chicks with these nutrients and supplements has been found to improve total digestive tract development, increase growth rate and feed efficiency, reduce post-hatch mortality and morbidity, promote growth of beneficial gut microbiota, improve the immune system and the response to enteric antigens, reduce incidence of developmental skeletal disorders, and increase in muscle development and breast meat yield. Further works are required to fine-tune the AS, SY, and JB wrote this review manuscript. BM reviewed the manuscript and provided critical suggestions and comments. RJ decided a review topic, reviewed the literature and this review manuscript and provided critical review, suggestions, and comments.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.


The cornea is a unique tissue and the most powerful focusing element of the eye, known as a window to the eye. Infectious or non-infectious diseases might cause severe visual impairments that need medical intervention to restore patients' vision. The most prominent characteristics of the cornea are its mechanical strength and transparency, which are indeed the most important criteria considerations when reconstructing the injured cornea. Corneal strength comes from about 200 collagen lamellae which criss-cross the cornea in different directions and comprise nearly 90% of the thickness of the cornea. Regarding corneal transparency, the specific characteristics of the cornea include its immune and angiogenic privilege besides its limbus zone. On the other hand, angiogenic privilege involves several active cascades in which anti-angiogenic factors are produced to compensate for the enhanced production of proangiogenic factors after wound healing. Limbus of the cornea forms a border between the corneal and conjunctival epithelium, and its limbal stem cells (LSCs) are essential in maintenance and repair of the adult cornea through its support of corneal epithelial tissue repair and regeneration. As a result, the main factors which threaten the corneal clarity are inflammatory reactions, neovascularization, and limbal deficiency. In fact, the influx of inflammatory cells causes scar formation and destruction of the limbus zone. Current studies about wound healing treatment focus on corneal characteristics such as the immune response, angiogenesis, and cell signaling. In this review, studied topics related to wound healing and new approaches in cornea regeneration, which are mostly related to the criteria mentioned above, will be discussed. Diseases affecting the cornea can be either infectious or non-infectious, and both may cause severe visual impairments requiring intervention. Trachoma, onchocerciasis, corneal ulceration, corneal dystrophies, and xerophthalmia are some of the major causes of blindness worldwide Sommer, . HoweverThe most striking advance in the medical treatment of corneal diseases over recent decades has been corneal transplantation and, nowadays, the cornea is the most commonly transplanted tissue worldwide. Corneal transplantations are divided into two main categories based upon the amount of surgically replaced tissue. In penetrating keratoplasty, the entire cornea is replaced with a donor tissue. However, in a newer procedure called lamellar keratoplasty, only the damaged layers are replaced with a donor graft, and the healthy part of the cornea is left intact. In lamellar keratoplasty, the integrity of the cornea and the surrounding tissues are preserved; as a result, better visual improvement is usually achieved. Unfortunately, there are sometimes poor outcomes because of graft rejection or late graft failure. Furthermore, according to a World Health Organization (WHO) report, 15–20% of patients who need corneal transplantation remain untreated because of the shortage of corneal donors , Osteo-Odonto-Keratoprosthesis (OOKP), AlphaCor, and the KeraKlear Artificial Cornea are the four types of keratoprostheses which have, so far, been commercialized. Over the last decade, implantation of artificial corneas has been dramatically increased. For instance, fewer than 50 units of B-KPro were implanted before 2002, while more than 9,000 implantations were carried out in 2014. In this section, recent advances in the design of artificial corneas will be discussed with regard to the type of materials employed.The B-KPro is the most widely implanted artificial cornea and was developed at the Massachusetts Eye and Ear Infirmary. There are two types of B-KPro each with different indications. The B-KPro type I is commonly used in patients with a non-cicatrizing disease such as repeated allograft failure, corneal opacity with extensive neovascularization, aniridia, trauma, etc. In contrast, B-KPro type II is used in cicatrizing diseases and severe dry eye conditions, such as severe autoimmune ocular diseases. Both types of B-KPro have similar compartments but are different in some details. They have a front plate and a back plate, which act to sandwich a fresh donor cornea; a titanium locking-ring is used to secure the plates. Medical grade polymethyl methacrylate (PMMA) with the ability to block UVA/UVB is used for the front and back plates. The idea of using PMMA was conceived due to its ability to induce only minimal inflammatory responses in the eye  is an alternative approach. Multiple efforts in using more biologically compatible substances resulted in the introduction of OOKP in 1963 by Strampelli  (PHEMA). The central core of the device is a transparent PHEMA cylinder, and the skirt is a porous PHEMA material to improve biointegration with the surrounding corneal stromal tissue. Nevertheless, the rejection rates of the synthetic skirt is much higher than the biological skirts discussed above  has introduced a newer design to address the limitations of previous keratoprostheses while retaining their advantages. KeraKlear Artificial Cornea is a one-piece keratoprosthesis without any need for a donor cornea. Moreover, using only acrylic material allows KeraKlear to be foldable and injectable. However, its implantation is technically challenging, and further investigations are needed to overcome these challenges Pineda, . BesidesCorneal wound healing, like wound healing in general, is a complex and dynamic process which is divided into four phases: the hemostasis, inflammation, cell proliferation, and remodeling phases. In fact, wound healing involves the interaction of various different cell lineages and a choreographed series of cellular events resulting in the replacement of the missing tissue or cellular structures. The cornea has special characteristics, which are important to consider in choosing a treatment strategy for wound healing. The most prominent characteristics of the cornea include its limbus zone, its lack of blood vessels, and its immune privilege see . As a reGenerally, the transparency and avascularity of the cornea are essential for proper vision. The main factors, which are threatening the corneal clarity are inflammatory reactions, neovascularization, and limbal deficiency. As a result, avoiding angiogenesis, maintaining immune privilege, and supporting the limbus zone are strategies that have been investigated to combat processes that endanger corneal transparency. Immune privilege describes the natural lack of inflammation in the cornea, but when wound healing is taking place, the immune response needs to be modulated and limited. The influx of inflammatory cells causes scar formation and destruction of the limbus zone. Since there is a naturally enhanced production of proangiogenic factors during wound healing  play a pivotal role in wound healing by carrying out intercellular signaling and communication with stromal cells. Exosomes are cell-derived nanoscale vesicles containing bioactive molecules, which mediate intracellular signaling , Fibroblast growth factor-basic (FGFb), Hepatocyte growth factor (HGF), and TGF-β1) released from the AM preparation had a complex, possibly non-linear effects, on HCECs. In another study Ogawa et al. identified an active matrix component [Heavy chain-hyaluronan/pentraxin3 (HC-HA/PTX3)] that was shown to exert the anti-inflammatory and anti-scarring effects of AM. Their studies revealed that subcutaneous and subconjunctival injection of HC-HA/PTX3 might be a novel approach in the treatment of ocular disease , CD31, and alpha-smooth muscle actin (α-SMA) expression.Anti-inflammatory agents like corticosteroids have long been topically applied for the treatment of ocular inflammation. However, they show a low therapeutic efficacy in the treatment of neovascularization, because of their poor corneal permeability, and lack of bioavailability  combined with an essential fatty acid , VEGF, TGF-β, HGF, and tumor necrosis factor-beta (TNF-β) in the wound healing process. Among these growth factors, PDGF, TGF-β, and HGF played a pivotal role in modulating cell proliferation and myofibroblast differentiation. There is extensive literature available concerning the role of growth factors in corneal wound healing  are essential in the maintenance and repair of the adult cornea as they support the repair and regeneration of corneal epithelial tissue. In fact, this undulated limbal region is considered to be a niche for LSCs, which play a critical role in the corneal wound healing process. In limbal tissue engineering, like the other tissues, three factors affect the regeneration of injured tissues: cell availability, suitable and biocompatible scaffolds, and the presence of growth factors. In corneal disorders, depletion or the absence of LSCs results in impairment of the corneal wound healing process. Regarding tissue engineering, there are three approaches to remedy this deficiency. The first approach is cell transplantation using the desired cell population obtained in tissue culture. This approach has been considered as a type of corneal transplantation that was briefly discussed in the previous section. The second approach is cell transplantation using cells such as corneal epithelial, where stem cells are dissociated, cultivated on a supportive matrix (biosynthetic scaffold) like an amniotic membrane, fibrin gel or polymers, and then injected into the desired location in the cornea. The main problem in the last approach is the lack of stem cell enrichment because the stem cells which are used for transplantation contain heterogeneous cell populations. This deficiency might result in a graft failure , and the use of a contact lens have gained more attention as they reduce the risk of xenobiotic infections and side effects from using non-Food and Drug Administration (FDA) approved biomaterials. Girolamo's group developed a novel autologous technique by using an FDA-approved soft contact lens as a carrier and bandage to protect the eye during LSC transplantation, thereby promoting corneal repair and regeneration  have been successful in the regeneration of different tissues (Koizumi and Okumura, “Form follows function” is a well-known concept accepted as a principle in the field of modernist architecture; although it is also universally observed throughout the nature. According to this principle, the shape of an object should be predominantly based upon its intended function or purpose (Griffith and Harkin, From the earliest concepts such as replacement of the opaque cornea, to cornea wound healing and regeneration, ophthalmologists, and material scientists worldwide have faced a variety of challenges. Advances in visualization techniques and histology have made significant progress in the fundamental understanding of cornea structure and its microenvironment. Due to this valuable information and nanotechnology advances, therapeutic strategies in devastating corneal diseases have turned from corneal replacement into corneal wound healing and regeneration (Kargozar and Mozafari, Most of the suggested treatments have shown promising results for wound healing at the ocular surface, and entire thickness dystrophies were neglected. While, in order to reduce transplantation of a donor cornea, tissue engineering of the whole thickness of the cornea must be considered. Corneal stromal and endothelium tissue engineering have recently shown noticeable progress (Matthyssen et al., in vivo confocal fluorescence microscopy, and full-field optical microscopy are part of visualization techniques which help to quantify corneal architecture (Grieve et al., Until now, most studies have worked on introducing new materials and biochemical approaches in cornea wound healing and regeneration; while paying attention to physical properties of these approaches might be a leap in this area. For instance Long et al. have tried to use a cross-linking agent in collagen membrane to regulate collagen fibril spacing and therefore improve optical clarity of collagen and increase permeability of neurites (Long et al., Considering the remarkable role of stem cells in tissue regeneration, a large part of future studies is expected to focus on the deployment of stem cells on cornea wound healing and regeneration (Rahmati et al., MoM and RA wrote the first draft. SO and MG wrote the first draft and analyzed the data. FM and MaM, created the idea, managed the team, and finalized the draft.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.