Articular cartilage has a low self-repair capacity due to the lack of vessels and nerves. In recent times, nanofiber scaffolds have been widely used for this purpose. The optimum nanofiber scaffold should stimulate new tissue's growth and mimic the articular cartilage nature. Furthermore, the characteristics of the scaffold should match those of the cellular matrix components of the native tissue to best merge with the target tissue. Therefore, selective modification of prefabricated scaffolds based on the structure of the repaired tissues is commonly conducted to promote restoring the tissue. A thorough analysis is required to find out the architectural features of scaffolds that are essential to make the treatment successful. The current review aims to target this challenge. The article highlights different optimization approaches of nanofibrous scaffolds for improved cartilage tissue engineering. In this context, the influence of the architecture of nanoscaffolds on performance is discussed in detail. Finally, based on the gathered information, a future outlook is provided to catalyze development in this promising field. There are three forms of cartilage in the body; hyaline or articular cartilage, fibroelastic cartilage (meniscus), and elastic cartilage Cartilage architecture predominantly comprises collagen type II, VI, IX, X, chondrocytes, and glycosaminoglycans. Collagen type II mainly forms 90-95% of the fiber network of the extracellular matrix of cartilage and provides its cartilaginous framework and tensile strength AC is divided into three areas: superficial, middle, and deep, each of which has its distinct properties; the number of cells, shape, size, and direction of collagen fibers and proteoglycans Figure  14-16. TAC has a low post-traumatic injury or wear self-repair capacity due to a lack of vessels and nerves. Furthermore, its cell proliferation capacity is not satisfactory Treatment of AC injuries is one of the most challenging issues of musculoskeletal medicine due to the poor intrinsic ability of this tissue to repair itself. Nevertheless, restoring AC is essential as it can relieve pain and improve function. Most importantly, it can delay or prevent the onset of arthritis. The most common procedures for cartilage restoration are as follows.Microfracture is commonly used for cartilage restoration by marrow stimulation. This method increases the migration of mesenchymal stem cells (MSCs), the influx of growth factors, and platelets from bone marrow to the defect site. However, this method results in the formation of fibrocartilage rather than normal hyaline AC This therapeutic approach creates holes in the subchondral bone and supplies blood flow into defects to induce the repair of cartilage. The holes are made with a surgical drill or wire. Although the method is similar to microfracture, in this technique, the heat of the drill damages the AC tissues. It again results in the formation of fibrocartilage Abrasion Arthroplasty removes some areas of the AC to create new capacities for joint surfaces. Hence, it can eliminate the injured cartilage tissue Autologous Chondrocyte Implantation (ACI) is used to repair AC defects in a surgical approach. ACI is divided into a two-step procedure. In the first surgical procedure, healthy cartilage tissue is taken from a non-weight-bearing area of the joint during an arthroscopic procedure. The cartilage tissue is then sent to the laboratory, and chondrocytes are isolated, which are subsequently cultured and proliferated. The second surgical procedure (arthrotomy) is an open procedure in which the newly grown cells are injected into the articular cartilage defects OCA has demonstrated consistent clinical results and can be used to treat a variety of articular defects of the knee using size-matched cadaveric donor plugs that permit immediate structural restoration of the joint articular surface All described treatment strategies have been applied to regenerate cartilage lesions to restitute articular function and relieve the associated pain. Although they decrease patient discomfort and enhance joint mobility, the repaired tissue is often fibrocartilage with less clinical action in vitro and is used to test the biocompatibility of the materials Tissue engineering is used to reconstruct and regenerate damaged tissues. It is a medical technology that has attracted considerable attention in recent times. The main purpose of this approach is not only to repair tissue but primarily to improve organ function. Tissue engineering also has diagnostic applications made Since the current surgical procedures for regenerating a cartilage defect have considerable drawbacks, 3D scaffolds might offer promising results to facilitate the restoration of target tissues in vitroOn the one hand, synthetic polymers are reproducible, and their properties can be easily controlled Regardless of the origin, the nanofiber scaffolds should stimulate the growth of new tissue in all aspects and mimic the characteristics of AC and the area below the articular cartilage. In addition, the scaffold should exhibit the potential to merge with the target and withstand physical activity tissue Scaffolds have been applied to improve tissue regeneration by recapitulating a difference in the physical structure of the tissue. Different strategies have been used, including fibrous self-synthesis, phase segregation, and electrospinning of nanofibers to produce the fibrous collagen network of the extracellular matrix via the aid of nanotechnology. Today, in reconstructive medicine, electrospinning scaffolds are used to repair and regenerate the tissues, such as heart muscle, cartilage, bone, nerve, and others. Interestingly, optimization of the nanoscaffold synthesis conditions may even lead to fiber alignment to obtain anisotropic materials for cell growth Figure  61.Some research has shown the potential of electrospinning of fiber scaffolds for cartilage tissue restoration In another study, the culture of MSCs on electrospun PCL nanofiber stimulated the fibroblast-like morphology. In contrast, the dynamic condition led to a round-shaped morphology with elevated collagen type I, II, and sGAG Other hybrid nanofiber scaffolds have also been applied in this context. For instance, PLGA/collagen scaffolds have shown optimal chondrogenesis potential Although nano-sized structures simulate well the ECM components, they can also promote cell spreading and restrict infiltration The synthesis of 3D nanofibrous scaffolds has also highly enhanced the chondrogenesis of different SCs Research has also shown that scaffolds with parallel fibers and random Poly(l-lactide) (PLLA)/Polycaprolactone (PCL) Although nano- and microfibers electrospun scaffolds maintain and support the growth and proliferation of bone marrow-derived stem cells, nano-fibrous PCL scaffolds play a greater cartilaginous activity. That was shown by produced sGAG and collagen type II expression, and this scaffold may be suitable for the repair of the superficial area of the scaffold. Other studies have reported similar results with nanofiber scaffolds, as opposed to microfiber scaffolds or smooth (film) Poly(L-lactide) (PLLA) In swine, as a large animal, the implantation of PCL/BMSCs in full-thickness cartilage defect of AC led to a complete repair and formation of hyaline cartilage-like tissue 6 months after seeding in vivo and the formation and design of nanofiber scaffolds are currently hot topics in the tissue engineering field because of its merits. Still, the examined literature reveals increasing demand for qualified tissue regeneration strategies to increase the number of successful transplantations. Thus, it is vital to develop strategies for creating cells, tissues, and organs with good compatibility to reduce the future rejection rate of clinical programs This review discussed the application of nanofiber scaffolds that can result in the complete synthesis of native hyaline cartilage tissue in optimum situations. Tissue replacement through non-biological and regenerative systems Importantly, there is much room to optimize the performance of nanoscaffolds. For instance, future scaffold constructs will only be successful if improved compatibility regarding the ECM is realized in vivo, more effort is still necessary for shifting the information from in vitro to in vivo phase. Only then this promising concept will serve society and improve the quality of our life.In this context, the fabrication and implantation of appropriate bioreactors that eventually aid in more ECM formation and the achievement of artificial constructs mimicking native cartilage tissues should be considered. Finally, although several investigations produced promising results regarding the efficacy of nanofibers for repairing cartilage defects


Here, we reviewed the application of microfluidic systems in modeling TME and the potential of these devices in predicting and monitoring immunotherapy effects.With continued advances in cancer research, the crucial role of the tumor microenvironment (TME) in regulating tumor progression and influencing immunotherapy outcomes has been realized over the years. A series of studies devoted to enhancing the response to immunotherapies through exploring efficient predictive biomarkers and new combination approaches. The microfluidic technology not only promoted the development of multi-omics analyses but also enabled the recapitulation of TME  The tumor microenvironment (TME), as the soil of tumor growth and metastasis, is a complex and dynamic ecosystem mainly consisting of cellular components  and noncellular components , vascular networks, cytokines, chemokines, etc.). The stromal cells including but not limited to endothelial cells, mesenchymal cells, and fibroblasts are crucial in facilitating and sustaining tumor cells. Within the TME, tumor cells mostly communicated with other cells through ECM and the secretion of molecules such as cytokines, growth factors, and lipid mediators . The varThe complex and vital role of TME in oncogenesis and tumor progression promoted a significantly increased number of relevant research, in which the multi-omics analysis showed promising advantages in recognizing the complexity of TME and tumor immunological heterogeneity. The multi-omics data analysis was also adopted to investigate the correlation between the genetic or epigenetic characteristics and the TME infiltration pattern in lung adenocarcinoma (LUAD) based on The Cancer Genome Atlas (TCGA) database, and successfully constructed a significant prognostic model . AdditioWith the increasing evidence revealing the significance of TME in regulating tumor progression and the response to anti-tumor therapy, immunotherapies targeting TME have been widely expanded these years . The devin vitro, have been widely used in modeling the TME of different tumors. Therefore, microfluidic technology promoted research on optimizing tumor immunotherapy furtherly based on the various microfluidic tumor models.Nonetheless, the effect of immunotherapy is still unsatisfactory as the increased resistance and the comparatively low response of patients to these treatments . Thus, aMicrofluidics is a rapidly developed technology that made it possible to manipulate fluids flowing in channels of tens to hundreds of micrometers in size . With thThe manufacturing of microfluidic devices generally begins with using photolithography to create the mold, and then PDMS or other alternative materials were poured into the mold and cured, finally to form a PDMS microfluidic device with hollow microchannels. The unique microfluidic channels enabled the precise manipulation of flow such as mixture and separation of tiny fluids, chemical reactions, and microanalysis, which made the microfluidic chips attractive in the screening of rare cells, gene sequencing, separation and analysis of single-cell, extraction and purification of information RNA and so on. In this regard, microfluidic technology exhibited great potential in single-cell-omics analyses. For instance, an integrated proteomics chip (iProChip) based on microfluidic technology was designed and coupled with data-independent acquisition (DIA) mass spectrometry (MS) for the in-depth microproteomics identification and quantification . This miin vivo physical conditions such as vascular perfusion, air-liquid interfaces, shear stresses as well as the physical and chemical gradients  revealed the great potential of microfluidic technology in cancer metastatic research.Given the unique properties of the microfluidic system in recapitulating the structure, function, physiological and pathological characteristics of human tissues and organs, these devices have been widely used to effectively mimic and analyze tumor microenvironment in vitro, and furtherly explored the effect of interactions between ECM and fibroblast on cancer invasion (via inducing the expression of glycoprotein non-metastatic B (GPNMB) on cancer cells in the 3D microenvironment. Another 3D culture model, integrated 3D tumor spheroids (TSs), and CAF on a microfluidic chip was used to recapitulate the interaction between TSs and fibroblasts (in vitro model.An increased number of studies are focused on the application of microfluidic technology in modeling the extracellular matrix (ECM) of TME which could promote the invasion, migration, and metastasis of cancer. For example, a microfluidic device created via the microfluidic called LumeNEXT was adopted to mimic the breast cancer TME invasion   cells within the central channel (via the side channel. It was found that NK cells presented high cytotoxicity in consensus molecular subtypes1 (CMS1) CRC cells in this tumor vasculature model. This platform contained 28 wells that allowed performing various high-throughput experiments ranging from interactions of immune and cancer cells within TME to drug screening for immunotherapy. A network platform with interconnected microfluidic channels was created to mimic a highly vascularized system. This novel microfluidic platform imitated the interactions between tumor cells and vasculature, and succeed in modeling vessel leakiness presented in the TME , lacking basement membrane, are leakier than blood vessels, promoting the metastasis of cancer cells. To decipher the influence of ECM on the lymphatic vessel (LV) in Bca, a microfluidic device was designed to generate LVs within a collagen hydrogel  Lugo-Ci. This miMicrofluidic models have also exhibited significant advantages in recapitulating the interplays between cancer and the immune system. A tumor-on-chip platform allowed the interplay of cellular and non-cellular components, modeling the TME and exploring the influence of TME on immune cell recruitment . It was in vitro microfluidic systems. The probable effect of immunotherapy on head and neck squamous cell carcinomas (HNSCC) patients was determined using an in vitro 3D microfluidic chip, which loaded different ICIs, Indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitors, and PD-L1 antibodies  or patients (PDOTS)  . The resresearch . With thresearch  (Brionesresearch . To enharesearch  (Aboulkhresearch . The resresearch  (Cui et research .in vitro models. As the traditional 2D cell culture and murine model could not recapitulate the faithful TME of humans, microfluidic devices, which enabled mimicking the dynamic and complex TME, have become more and more attractive in creating the in vitro tumor models.The critical role of TME in mediating tumor progression and affecting therapeutic outcomes is more and more apparent with the increasing evidence from plenty of research. From this, various therapies targeting the various components of the TME have been developed, and some therapeutic drugs have improved patient prognosis to some extent . Among tin vitro (in vitro (in vitro model (With the continuous progression over the past years, microfluidic devices, based on microfluidic technology, have been developed to recapitulate the physiological and pathological condition of humans in vitro . Microflin vitro . As for in vitro . By the in vitro . Furtherin vitro . The intin vitro , has beein vitro . Accordiin vitro . In thisro model  (Aref etro model .In contrast with conventional methods, microfluidics provides a more rapid and cost-effective technic to construct more controllable and reproducible methods for drug delivery and screening . The druAlthough microfluidic devices have exhibited comparable advantages over traditional 2D culture systems for modeling the TME in preclinical research, there are some challenges to further application for these devices. For instance, PDMS, the primary material of microfluidic devices, has been demonstrated to absorb small molecules which could affect the drug screening studies outcomes . While aMicrofluidic devices possess the huge potential to serve as a predictive and effective tool for immunotherapies to optimize the treatment of tumor patients. As CTCs and exosomes isolated using the microfluidic-based device have shown specific significance in the prediction and monitoring of response to ICIs, it is expected to produce an integrated microfluidic-based device that allowed high-throughput and automated assays for isolation and diagnostic test for patients. In the future, the breakthrough of microfluidic technology may simplify the design and manufacture of microfluidic systems, enable the minimization of these devices, and promote their application in clinical diagnosis and treatment.in vitro. We also discussed the challenges and future of microfluidic devices in clinical application. Overall, we are looking forward to microfluidic systems that can 1 day realize their significance in clinical practice and promote cancer immunotherapies and precision medicine.In summary, we briefly introduced the microfluidics including the application in multi-omics analyses, and then focused on the microfluidic technology applicated in the TME modeling, finally reviewed the potential of microfluidic systems for further application in immunotherapies according to their capacities in recapitulating the TME


Improvement in pesticide application and efficiency structure has long been recognized as having great significance in reducing pollution, ensuring food safety, and promoting green agricultural development. Based on theoretical analysis, using the survey data of 766 farmers in key tea areas in Shaanxi, Sichuan, Zhejiang, and Anhui provinces in China, the study empirically analyzes the influence of market incentives and livelihood dependence on farmers’ multi-stage pesticide application behavior. More specifically, the study employed ordered probit analysis to craft its findings. The dependent variable of this study is the multi-stage pesticide application problem of farmers, and the core independent variables are market incentives and livelihood dependence, and the judgment is based on the core variable coefficients of the econometric model of farmers at each stage. The study found the following: (i) Market incentives significantly prompted some farmers to give up synthetic pesticide application and farmers tend to choose green pesticides in the type of pesticide application. (ii) Livelihood dependence meant that the proportion of tea income significantly prompts farmers to apply pesticides, and also creates a tendency for farmers to choose green and low-toxic pesticides in the type of pesticide application. The planting period tends to have a moderate impact on applying green and low-toxic pesticides. (iii) The interaction term of market incentives and the proportion of tea income has no significant impact on farmers’ multi-stage pesticide application behavior. The interaction term of market incentives and planting years has impacted negatively on whether farmers apply pesticides, and has no significant impact on farmers’ choice of pesticide application types, but makes farmers increase the amount of green and low-toxic pesticides. (iv) The education level of the household head significantly promotes farmers to choose green and low-toxic pesticides. Seemingly, the brand effect of pesticides significantly encourages farmers to choose green and low-toxic pesticides. In external support, technical training significantly encourages farmers to choose green and low-toxic pesticides. Furthermore, better infrastructure and local market conditions significantly encourage farmers to reduce the use of conventional pesticides. Ever-increasing trends in the population and demands for food and fiber foster the chemical interactions in agriculture. However, most developing countries rely substantially on intensifying production, which eventually lowers the land quality and reduces the resistance to pests and other diseases ,2,3. PesWith the improvement in the market system, technological progress, and consumption upgrade, the impact of market incentives on farmers’ production behavior has become increasingly prominent ,17,18. MThe existing results provide a good reference for the study, but it can still be expanded on in the following two ways: First, though research on farmers’ pesticide application behavior based on market incentives or livelihood dependence has gradually increased, there is a lack of studies that integrate these two major factors in an integrated framework. Moreover, the regulatory role of livelihood dependence on market incentives and farmers’ pesticide application behavior has not been paid much attention by the existing studies. Second, the existing articles only study whether farmers apply pesticides, the types of pesticides or the number of pesticides applied, and the multi-stage pesticide application behavior in the decision-making process of whether farmers apply pesticides, selection of pesticide types, and selection of pesticides have not been clarified yet. Given this, the study divides the pesticide application behavior of farmers into multiple stages of whether to apply pesticide, selection of pesticide type, and selection of pesticide amount. The impact of market incentives and livelihood dependence on farmers’ pesticide application behavior was comprehensively analyzed, to provide a decision-making reference for the government to formulate relevant policies to promote farmers’ pesticide reduction, structure optimization, and agricultural green transformation.Existing studies .Market incentives have a significant impact on farmers’ multi-stage pesticide application behavior.For intensifying agricultural production, the impact of livelihood dependence on farmers’ behavioral choices is generally considered from the perspective of household income and consumption ,42. The The second is the planting period. Farmers will accumulate experience and experience in the process of tea planting and become an important component of their human capital and family culture  and prevHypothesis 2 (H2).Subsistence dependence has a significant impact on farmers’ multi-stage pesticide application behavior.As discussed above, market incentives affect farmers’ multi-stage pesticide application behavior through a series of mechanisms, and livelihood dependence may moderate the impact of market incentives on farmers’ multi-stage pesticide application behavior. First, in the face of production pressure, technical pressure, and quality pressure under market incentives, income dependence has an important impact on farmers’ production choices ,59. On tHypothesis 3 (H3).Subsistence dependence has a moderating effect between market incentives and farmers’ multi-stage pesticide application behavior.The data in this study come from the research group’s survey data of farmers in 8 key tea areas in Shaanxi, Sichuan, Zhejiang, and Anhui from July to September 2021. First, the research team selected important tea-producing areas in Zhejiang, Sichuan, Shaanxi, and Anhui provinces for research based on tea production and economic development. Next, we randomly selected 3 townships from each county; after that, 2 administrative villages were randomly selected from each township. Finally, 10 to 20 farmers were selected by random sampling in the village. The survey was mainly conducted by questionnaire survey combined with interviews and obtained information such as farmers’ pesticide application behavior, household head characteristics, basic family situation, and organizational support. After excluding invalid questionnaires, a total of 766 valid questionnaires were obtained. The study employed ordered probit analysis to test the model, which is widely used by similar studies  distribution.In Formula (1), it To estimate the impact of market incentives and livelihood dependence on farmers’ choice of pesticide application types, the following probit model was constructed:N  distribution.In Formula (2), it To estimate the impact of market incentives, livelihood dependence, etc. on farmers’ choice of pesticide application rate, an ordered probit model for farmers’ selection of pesticide application rate was constructed. The equation is shown in Equation (3):In Formula (3), it Three variables were used to characterize the multi-stage pesticide application behavior of farmers. To ensure the validity of the data, after the investigators asked farmers whether to apply pesticides, they ensured that the farmers determine the type of pesticide application and the amount of pesticide application in the form of explanations. As shown in Market incentives, as a combination of price signals and product quality grades, have an important impact on farmers’ pesticide application behavior ,73. TherFarmers’ multi-stage pesticide application behavior is closely related to farmers’ interpersonal characteristics, family characteristics, land endowment, and affect cognition. Referring to the relevant literature  Control variables. Among household characteristics, the level of household income significantly encourages farmers to increase the amount of green and low-toxic pesticides. With the increase in household income level, farmers can afford the cost of green and low-toxic pesticides and tend to use green and low-toxic pesticides to replace labor. Among the garden land endowments, the area of tea gardens significantly encourages farmers to reduce the use of green and low-toxic pesticides. With the increase of scale, on the one hand, the factor allocation of farmers who choose green and low-toxic pesticides presents economies of scale, and the overall pesticide application level is better than that of small-scale farmers; Substitute, thereby reducing the application rate of green low-toxic pesticides. In pesticide cognition, the effect of pesticide brands significantly encourages farmers to reduce the amount of green and low-toxic pesticides. Tea sales are greatly affected by the brand of the place of origin. Therefore, farmers who choose low-toxicity will reduce the amount of green and low-toxic pesticides when they realize the negative effects of pesticides on the brand of the place of origin.The robustness test was carried out based on Model 1–Model 8. Specifically, the logit model was used to replace the probit model of Model 1–Model 4. The logit model replaces the ordered probit model of Model 4–Model 8, thereby obtaining Model 9–Model 16. It can be seen from Promoting the reduction in pesticide use for farmers and the optimization of the application structure are the key issues for reducing pesticide pollution, ensuring food safety, and promoting the green development of agriculture . The notHowever, in previous studies, scholars have mainly studied pesticide application by farmers from the aspects of policy incentives ,92, infoThe results show that market incentives are an important external driving force affecting farmers’ pesticide application behavior, while subsistence dependence is an important internal force affecting farmers’ pesticide application behavior. In the review study on integrated pest management practices in Europe, Lefebvre et al.  found siBased on the data of 766 farmer household surveys in key tea areas in Shaanxi, Sichuan, Zhejiang, and Anhui provinces, we empirically analyzed the impact of market incentives and livelihood dependence on farmers’ multi-stage pesticide application behavior. The study found that first, market incentives significantly prompted some farmers to give up pesticide application, but also created a tendency for farmers to choose conventional pesticides in terms of pesticide application types; market incentives had no significant impact on farmers’ conventional pesticide application rates but prompted farmers to reduce green low levels. Second, in the livelihood dependence, the proportion of tea income significantly prompts farmers to apply pesticides, and also creates the tendency for farmers to choose green and low-toxic pesticides in the type of pesticide application, but it has no significant impact on the application rates of farmers with conventional pesticides and green and low-toxic pesticides. The planting year has no significant effect on whether farmers apply pesticides, but it creates the tendency for farmers to choose conventional pesticides in the type of pesticide application; the planting year makes the farmers choose to increase the number of conventional pesticides, and has no significant impact on the amount of green and low-toxic pesticides for farmers. Third, the interaction term of market incentives and the proportion of tea income is not significant; the interaction term of market incentives and planting years prompts farmers to choose pesticides, which has no significant impact on the types of pesticides applied by farmers but makes farmers increase the amount of green and low-toxic pesticides.Based on the above conclusions, the following implications are drawn. First, strengthen market incentives to ensure the effective transmission of market signals of high quality and good prices. The government should give financial support to market organizations, and provide policy financing for the purchase of necessary inspection equipment, technology introduction fees, etc., or preferential policies such as tax deductions, to improve the ability of market organizations to identify product toxicity; build an agricultural product information data platform to improve the quality of agricultural products. The traceability system enhances the transparency of agricultural product quality information and ensures the effectiveness of market incentives. Second, it provides economic incentives for high-standard pesticide application farmers, strengthens technical training, and implements the upgrading and transformation of old tea gardens and special training projects for old tea farmers. On the one hand, the government should set up a special fund to provide physical or financial support to farmers who use green and low-toxic pesticides and reduce the number of pesticide applications, to reduce farmers’ operating costs and potential risks. On the other hand, the government should increase the knowledge and skill training on pesticide application for farmers through technical training, farmer field schools, etc., to weaken the misunderstanding of planting years, effectively improve farmers’ pesticide skills, and design special projects to upgrade old tea gardens. This gradually reverses the irrational pesticide application behavior of farmers. Third, it formulates differentiated guidance strategies based on fully grasping the relationship between market incentives and livelihood dependence and its impact on farmers’ pesticide application behavior, such as establishing a market credit system, issuing product quality awards, or honorable household awards, financial subsidies, and technical training, to create conditions for farmers with different livelihoods to adapt to market incentives, adapt to large market demands, and green transformation.In the future, this study can be expanded within the aspects of the connotation and types of market incentives, include more psychological and government regulation variables and build a more realistic decision-making scenario. Moreover, potential studies should focus on deeply revealing the impact of market incentives and livelihood dependence on farmers’ multi-stage pesticide application behavior. The study only focussed on four provinces while future studies can expand their target study area to grasp the whole scenario of the country. The study only explored limited control variables. However, the potential studies should include several crucial control variables which may have certain impacts such as the degree of adopting environmentally friendly pest management tactics. Moreover, the descriptive statistics should be explored more critically and comprehensively according to the local conditions and socio-economical circumstances.


Estimation of metabolites concentrations has proven beneficial in several clinical conditions, including age-related neurodegenerative disorders, and normal physiological brain aging. In healthy and pathological brain aging, significant physiological changes include cortical atrophy, impairment of bloodflow and glucose metabolism, and elevated inflammation; which eventually lead to more severe structural but also functional and behavioral changes. All of these root causes for physiological brain aging have a direct impact on neurochemistry; concentration levels of metabolites thus become key proxy markers for such physiological modulations. Taken together, this positions modern MRS as an extremely valuable imaging modality to assess neurological state, but also the therapeutic impact of a large range of interventions. In this Research Topic, we sought to collect novel contributions to the field from MRS researchers working either directly on the use of MRS and additional imaging modalities in the study of brain aging, or in development of MRS imaging in the perspective of furthering research in brain aging.In the past 20 years, proton magnetic resonance spectroscopy (MRS) has developed into an established and robust imaging method to gather critical neurochemical information Vints et al. assessed the interaction between MRS metabolites obtained from several regions of interest and blood-derived peripheral inflammatory markers interleukin-6 and kynurenine. They show that elevated levels of kynurenine were associated with increased levels of choline and myo-inositol in the left somatosensory and dorsolateral prefrontal cortices, suggesting a relationship between peripheral and cortical neuroinflammation.A crucial issue in brain aging, and one that has arguably stoked much interest in MRS research, is inflammation and, incidentally, the term coined inflammaging. Normal age-related inflammation is considered one of the main factors for development of neurodegenerative disorders. In their paper, Krishnamurthy L. C. et al. found that by combining MRS with pseudo-continuous arterial spin labeling MRI, coupling of Glx (glutamate + glutamine) and cerebral blood flow was significantly lower in healthy older adults compared to young adults. This suggests a potential role for altered glial activation in older adults, and differential roles for Glx, GABA, and CBF in the study of the aging brain in the context of upper extremity motor performance. Further, the positive coupling between cardiorespiratory fitness (as quantified by VO2max) and metabolite concentrations GABA and Glx suggests that higher fitness allows for a more efficient metabolic shift that facilitates improved performance on cognitive-motor tasks.In another publication from the Research Topic, Krishnamurthy V. et al. investigated the age differences in performance of a language task. They found that, although resting levels of GABA and Glx did not predict task-BOLD amplitude, GABA related to the start of the task block that was less difficult while Glx related to the end of the task block that was more difficult. Importantly, they report that GABA and Glx may have a push-pull relationship with vascular factors  derived from task-fMRI, indicating that inhibitory and excitatory metabolites may influence neurovascular coupling differentially. This elicits the importance of combining neurovascular-natured BOLD and MRS in the study of aging, and shows that multi-modal assessments of the neurophysiology may help tease apart the neural and vascular contributions of the BOLD signal in an aging-related context.In another study combining MRS with task-based blood oxygen level-dependent (BOLD) MRI, Song et al. compared peak-fitting approaches to glutathione quantification with HERMES and MEGA-PRESS acquisition sequences. They show that linear combination modeling improved reliability of glutathione quantification for HERMES data, providing additional guidelines for future utilization of this very sequence.Besides the study of glutamate and GABA, metabolites like glutathione, the main antioxidant of the human central nervous system, are also of crucial interest in the study of physiological brain aging. Accordingly, novel approaches to reliably estimate levels of glutathione have been at the forefront of MRS technical developments in recent years. In their paper, Gu et al. have investigated data in several studies on MRS markers for early diagnosis of Parkinson's disease in subcortical areas substantia nigra and globus pallidus. They report that the ratio of n-acetylaspartate to creatine showed a significant difference between patients with early-stage Parkinson's disease and healthy controls. This suggests obvious neurochemical modulation in subcortical nuclei with depletion of dopaminergic neurons, but also that MRS could provide additional metrics in the evaluation and identification of early-stage Parkinson's disease.Finally, although this Research Topic has mostly focused on brain aging, it is important to mention the important body of work on neurodegenerative disorders which has been using MRS as main imaging modality. Importantly, some of those works have focused on the identification early biomarkers for Alzheimer's disease e.g., , 2], but, but2)],Taken together, the works presented in this Research Topic demonstrate recent advancements in MRS, and other imaging modalities, in the study of brain aging and related pathologies. Such advances advocate for (1) the relevance of MRS in the field and (2) the importance of the continuing effort in developing MRS in the study of the aging brain.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


As aging and cognitive decline progresses, the impact of a sedentary lifestyle on the appearance of environment-dependent cellular morphologies in the brain becomes more apparent. Sedentary living is also associated with poor oral health, which is known to correlate with the rate of cognitive decline. Here, we will review the evidence for the interplay between mastication and environmental enrichment and assess the impact of each on the structure of the brain. In previous studies, we explored the relationship between behavior and the morphological features of dentate gyrus glial fibrillary acidic protein (GFAP)-positive astrocytes during aging in contrasting environments and in the context of induced masticatory dysfunction. Hierarchical cluster and discriminant analysis of GFAP-positive astrocytes from the dentate gyrus molecular layer revealed that the proportion of AST1 (astrocyte arbors with greater complexity phenotype) and AST2 (lower complexity) are differentially affected by environment, aging and masticatory dysfunction, but the relationship is not straightforward. Here we re-evaluated our previous reconstructions by comparing dorsal and ventral astrocyte morphologies in the dentate gyrus, and we found that morphological complexity was the variable that contributed most to cluster formation across the experimental groups. In general, reducing masticatory activity increases astrocyte morphological complexity, and the effect is most marked in the ventral dentate gyrus, whereas the effect of environment was more marked in the dorsal dentate gyrus. All morphotypes retained their basic structural organization in intact tissue, suggesting that they are subtypes with a non-proliferative astrocyte profile. In summary, the increased complexity of astrocytes in situations where neuronal loss and behavioral deficits are present is counterintuitive, but highlights the need to better understand the role of the astrocyte in these conditions. Unhealthy brain aging and cognitive decline associate with a sedentary lifestyle and, at a cellular level, this is accompanied by astrocyte hypertrophy, myelin dysregulation, neurovascular dysfunction  and the Several experimental models of masticatory dysfunction have been explored to clarify the cellular and molecular mechanisms associated with memory impairment ,26,27. FAt the cellular level, cognitive decline has been linked to neuroinflammation via the enhanced activation of astrocytes, oligodendrocytes, and microglia ,36,37 anAs a function of environmental stimuli ,42, age In rodent models of dysfunctional mastication, induced either by tooth loss, raised bite or soft diet, cognitive decline is associated with differential effects on astrocytes in different areas and different layers within the same greater brain region ,57,58,59From optogenetics and chemogenetics studies, in which astrocytes can be selectively manipulated, emergent data has provided evidence that astrocytes directly participate in cognition ,67,68,69Here, we have re-evaluated morphological data concerning GFAP-positive astrocytes in the dentate gyrus at different ages under the influence of contrasting environments and induced masticatory dysfunction. We concluded that the differential effects of these challenges on astrocyte morphological complexities may reflect the existing transcriptomic diversity of astrocyte dentate gyrus phenotypes, and that environment and masticatory activity interact to alter the spatial distribution and morphology of glial fibrillary acidic protein in aged astrocyte arbors.It is well known that long-term voluntary running improves learning and memory, by enchancing the strength of neuronal connections, through synaptic plasticity in the hippocampus ,79, and The elements inside enriched cages provide novelty, visuo-spatial and somatomotor stimuli and social interaction, but the stimuli for neurogenesis and the release of neurotrophins originate from voluntary exercise . ComparaInnate behavioral and physiological programs ensure survival and must be flexible enough to cope with environmental changes and build adaptive responses ,92. The Oral and cognitive health are interconnected  and the Mice housed in standard cages have reduced physical fitness and impaired thermoregulation, which leads to decreased ethological behavior and welfare . In addiThe hypothalamic paraventricular nucleus (PVN), a high order integration center between the neuroendocrine and autonomic nervous systems, is affected by chewing, which reduces the number of corticotropin releasing factor positive cells inhibiting the autonomic releasing of adrenaline and noradrenaline via locus coeruleus . We previously examined, in an open field, the combined influences of contrasting environments and masticatory regimens on exploratory and locomotor activity and found that all mice, independent of the masticatory condition, environment, or age, exhibited a similar temporal organization of their spatial horizontal exploratory activity in the open field task. However, aged mice living in life-long environmental enrichment and with normal masticatory activity showed reduced tendency to avoid open/lit spaces and that a contrasting diet regimen—a reduction or reduction/rehabilitation of mastication—showed differential effects at different ages. The combined effects of aging, environmental impoverishment and reduction in masticatory activity affect the innate behavioral repertoire of mice to explore novel environments and to assess risk .As lifelong environmental enrichment , physicaIn animal models all the approaches that have been used to induce masticatory dysfunction  are associated with impairment of spatial learning and memory, a reduction of the number of hippocampal pyramidal neurons, the downregulation of brain derived neurotrophic factor, decreased synaptic activity, impaired neurogenesis in dentate gyrus and increased glial cell proliferation, which seem to be dose-dependent through the reduction of chewing-related stimuli  is more active than the horn of Amon (CA1-CA2-CA3) and the subiculum, and that in both the coding process and information retrieval, the rostral  pole is more active than the caudal  pole . In factThe entorhinal-to-dentate gyrus pathway is involved in memory formation carrying spatial and non-spatial information through the medial and lateral perforant excitatory pathways onto granule cells ,132. AstEvidence has now emerged in rodents that the astrocyte is an essential mediator of learning and memory  and thatIn the next section, we review our previous findings on learning and memory impairment induced by age, masticatory dysfunction and sedentary lifestyle and discuss the underlying mechanisms associated with differential effects on the morphological complexity of astrocytes at the molecular layer of the dentate gyrus.It is known that physical exercise promotes morphological changes in astrocytes, and astrocytes may contribute to episodic memory function ,140. AstHere, we re-evaluated our findings of mouse age-related cognitive decline and used astrocyte morphological complexity to disentangle the multivariate morphological changes that were induced . Our preMorphological complexity definition in the present report was adapted to astrocyte morphology from the definition of morphological complexity of neuronal dendritic arbors described elsewhere  as folloIn general, astrocyte arbors with the greater complexity phenotype (AST1) from an enriched environment, independent of masticatory regimen or age, showed thinner and more ramified branches than astrocytes from mice raised in an impoverished environment. This effect, however, is not readily recognized in astrocytes with the lower complexity phenotype (AST2). Thus, AST1 and AST2 morphological complexities are diversely affected by environment, aging and masticatory dysfunction, suggesting that astrocyte morphology does not respond linearly to these influences and that these morphotypes may have differential physiological roles.Astro-glial morphological atrophy and loss of function seem to be part of neuropathological changes of the aging brain , and astIt has been suggested that astrocytes exhibit two main phenotypes associated with a proliferative profile surrounding areas of damaged tissues and a non-proliferative, but reactive, profile retaining basic structural organization and cell interactions in intact tissues . All recAlthough dorsal and ventral hippocampal regions show similar laminar and cellular organization, their connectivity to other brain regions are different ,153,154.Astrocyte morphology is affected by aging ,171,172,We used a stereological sampling approach to select astrocytes from the molecular layer of dorsal and ventral dentate gyrus for three-dimensional reconstructions and the Hierarchical Ward’s Minimum Variance Clustering Method  was applThree main astrocyte morphotypes, exhibiting significant differences in morphological complexity mean values, were found in all experimental conditions. These phenotypes were differentially affected by dysfunctional mastication, sedentary lifestyle, and aging. A fourth astrocyte phenotype, with very low morphological complexity values, was found in 5 of 12 experimental conditions in the dorsal dentate gyrus and in only one instance the ventral dentate gyrus. Due to its asymmetric distribution in the experimental conditions and low occurrence, the comparative analysis of the 4th phenotype with the other phenotypes could not be undertaken and they were removed from our analysis. Data for the three main phenotypes from dorsal and ventral dentate gyrus are exhibited in In contrast to astrocytes reconstructed from the molecular layer of dorsal dentate gyrus, the influence of the masticatory regime on the mean value of astrocyte morphological complexity was more conspicuous in the ventral region. Indeed, all chewing regimes, regardless of environment and age, influenced the mean value of morphological complexity of AST1 and AST2 morphotypes. Interestingly, the AST3 subtype in the ventral dentate gyrus was influenced by age and diet regime, but it was not influenced by environment. A previous report, limited to search for age influence on morphological complexity of GFAP astrocytes, demonstrated remarkable heterogeneity in the age-related changes in distinct subfields and along the dorsoventral axis of the hippocampus and in the entorhinal cortex of C57Bl6 mice . These aRecently , cyclic Overall, we have reviewed the rapidly expanding literature on astrocyte behavior in homeostatic conditions to highlight how the cellular and molecular characteristics relate to behavior. In particular, we have focused on the cognitive decline aggravated by a sedentary lifestyle and by masticatory disorders by re-evaluating their influence on the morphology of the dentate gyrus astrocytes. We found that reduction of masticatory activity reduced learning rates at all ages, which was independent of the environment, and that masticatory rehabilitation and environmental enrichment recovered age-related memory impairments. These spatial learning and memory deficits, and their recovery by an enriched environment and masticatory rehabilitation, were associated with differential morphological changes of the dentate gyrus astrocytes. Unbiased selection of astrocytes for three-dimensional reconstruction from the external one-third of the molecular layer of the dentate gyrus, using a random and systematic stereological approach followed by hierarchical cluster and discriminant analysis, revealed the presence of four main astrocyte morphotypes. These morphological families, identified in the external third of the molecular layer of both dorsal and ventral dentate gyrus, were differentially affected by age, sedentary lifestyle, and masticatory dysfunction, which suggests that they have different physiological roles in homeostatic conditions. All the morphotypes retained basic structural organization in intact tissue, suggesting that they are indeed subtypes of a non-proliferative astrocyte profile. Future studies directly assessing astrocyte functions in the aging mouse brain should integrate molecular, cellular systemic and behavioral analysis to unravel the underlying mechanisms associated with aging cognitive decline aggravated by sedentary lifestyle and masticatory dysfunction.


PLOS Biology, Mercey and colleagues present optimized expansion microscopy for retinal tissue, which represents a huge step forward in our ability to study these blinding conditions.Being able to see the beauty of this world is a wonderful thing unfortunately unavailable to people with inherited blindness. In this issue of  This Primer explores a PLOS Biology study which uses ultrastructure expansion microscopy to study the inner scaffold of the photoreceptor connecting cilium, the location of multiple proteins implicated in inherited forms of progressive sight loss such as retinitis pigmentosa and Leber congenital amaurosis. The projection at the back of the eye can be compared to the way an image is created by a digital film camera. Instead of a digital sensor, the back of the eye is decorated with several layers of interconnected neurons, called the retina (https://webvision.med.utah.edu). The outermost layer of the retina, last reached by the light, consists of millions of light-sensitive cells, known as the photoreceptors [Vision is one of the most valuable senses of the human body, as it allows us to see the magnitude and beauty of this world. Seeing begins when rays of light enter the eye via the cornea, are refracted by the lens, and projected to the back of the eye  and Leber congenital amaurosis (LCA), the most frequent and most severe form of inherited blindness, respectively . DespiteMercey and colleagues now overcome this limitation by, for the first time, applying ultrastructure expansion microscopy (U-ExM) on retina tissue . By implThe physical expansion leads to a spatial distancing of the molecules, making them more accessible for antibodies, which could improve the antibody specificity and binding capacity, eventually resulting in better distinguishable protein localization patterns. Indeed, Mercey and colleagues were able to show distinct localization of several proteins within the tidily organized connecting cilium ultrastructure, thereby revealing the existence and the composition of the connecting cilium inner scaffold . This inTaking advantage of the possibility for double labeling with tubulin and CEP290 on the expanded retinas, Mercey and colleagues could show that CEP290 localizes along the full length of the connecting cilium outside of the microtubule doublets with a previously unidentified , 9-fold LCA5 gene, specifically localizes to this bulge region, in accordance with previous EM analysis [By identifying the connecting cilium inner scaffold as structural zipper, Mercey and colleagues identified a distinct region, directly above the connecting cilium, which they called the “bulge region.” They found that this region was lacking both the inner scaffold and the Y-links, resulting in more dispersed microtubule doublets . Lebercianalysis , with a analysis , highligSPATA7 mutant mice, as shown by cryo-electron tomography [To validate the idea of the connecting cilium functioning as structural zipper, Mercey and colleagues assessed the photoreceptor development of FAM161A mutant mice from early postnatal to adult stage . Indeed,mography . This phmography .CEP290 and SPATA7 mutant retinas will expand previous knowledge on neonatal mouse retinas at the level of Y-links, analyzed by EM and stochastic optical reconstruction microscopy (STORM) [It would be of major interest to investigate the interplay between the connecting cilium inner scaffold, the Y-links, the bulge region, and the microtubule doublets in context of various inherited blinding conditions. For this, the optimized U-ExM by Mercey and colleagues  can be a (STORM) . Further (STORM) ,11.The application of U-ExM on retinal tissue thus opens new directions in understanding the biology of photoreceptors and the etiology of inherited blindness. Similar applications to other neuronal tissues and organoid models may, in the future, undoubtedly prove to be equally valuable.


The scientific and medical communities are becoming more aware of the substantial relationship between the function of the central nervous system (CNS) and the state of the gut environment. Parkinson's disease (PD) is a neurodegenerative disorder that affects the nigrostriatal pathway in the midbrain, presenting not only motor symptoms but also various non-motor manifestations, including neuropsychiatric symptoms and gastrointestinal (GI) symptoms. Over time, our knowledge of PD has progressed from the detection of midbrain dopaminergic deficits to the identification of a multifaceted disease with a variety of central and peripheral manifestations, with increased attention to the intestinal tract. Accumulating evidence has revealed that intestinal disorders are not only the peripheral consequence of PD pathogenesis, but also the possible pathological initiator decades before it progresses to the CNS. Here, we summarized recent research findings on the involvement of the intestinal environment in PD, with an emphasis on the involvement of the intestinal barrier, microbiome and its metabolites, inflammation, and enteric glial cells Parkinson's disease (PD) is the second most common neurodegenerative disorder and is dominated by motor symptoms. The most common pathological signature in the PD brain is the intracellular inclusions containing misfolded and aggregated alpha-synuclein (α-syn), also known as Lewy bodies, resulting in the loss of dopaminergic neurons in the substantia nigra. An estimated 25-80% of PD individuals may have gastrointestinal (GI) issues, including constipation, nausea, dyspepsia, dysphagia, and excessive drooling , 2. ConsThe GI tract serves as a semipermeable barrier, allowing nutrients, ions, and water to be absorbed while also regulating host interaction with a vast range of food antigens and bacterial metabolites. Therefore, intestinal leakage in PD patients may be a critical early trigger leading to the onset and/or development of PD process. Lewy bodies appear in the enteric nervous system (ENS) prior to the CNS , 5. BraaThe intestinal barrier consists of intestinal microbiota and the outer mucous layer, as well as the epithelium and lamina propria . The intThe loss of TJ protein increases intestinal permeability in the context of intestinal inflammation, according to the prevalent theory of "leaky gut" . Fig. 1Fig. 1. FTo date, studies attempting to assess intestinal permeability in PD patients have yielded only initial and contradictory findings. Davie et al. first diIndeed, PD is associated with increased intestinal permeability, which may be considered a contributing factor to the development of pathology. The alteration of intestinal permeability in the context of disease may be attribute to the microenvironmental regulation of intestinal barrier function, particularly ENS.Changes in intestinal microbiota, of course, may play a significant role in the altered intestinal permeability of PD patients, as there is convincing evidence indicating the interactions between intestinal microbiota and intestinal epithelial cells (IECs) . The ratBacterial components including lipopolysaccharide (LPS) or amyloid protein curli have been demonstrated to increase α-syn aggregation, lending credence to the idea that the gut microbiome is involved in PD . Kelly eIntestinal bacteria and their metabolites, primarily SCFAs, contribute to preserving the integrity of the IEB through modulating cell proliferation and differentiation, TJ protein expression, and mucosal permeability . In the In addition, the intestinal epithelium, being a mucosal tissue, continuously generates mucus and is covered with mucus, which serves as the first line of protection against pathogens. Mucus is generated by goblet cells and is made up of glycosylated mucin proteins as well as other protective compounds that aid in epithelial restoration. Microbiota products influence the production of intestinal mucus. Butyrate generated by benign microbiota components stimulates greater mucin secretion, forming a positive feedback loop for the preservation of the mucous barrier and its colonization by butyrate-producing commensals .Reduced levels of SCFAs in the colon may impede colonic motility, resulting in constipation in PD . In a stTJ protein synthesis is influenced by intestinal bacteria, and several pro-inflammatory cytokines generated by activated immune cells act on TJs to increase barrier permeability . InflammForsyth and colleagues  discoverEnteric glial cells (EGCs) are a unique type of peripheral glial cell that are distributed in the intermuscular, submucosal plexus, and extra-ganglionic regions like the muscular layer and mucosal lamina propria . EGCs haMany researchers have found that EGCs respond to damage activation primarily via the Toll-like receptors-2 (TLR-2) and Toll-like receptors-4 (TLR-4) . TLR-4 kDavid et al. hypothesized that altered expression of glial markers and pro-inflammatory cytokines in the colon of PD individuals can regulate the integrity and enhance the permeability of IEB. Although glial heterogeneity has been confirmed in the intestine, there are various physiological glial markers, including Sox-10, S100β and glial fibrillary acidic protein (GFAP), which fluctuate dynamically depending on the condition of the intestinal mucosa . The expEGC-driven neuroinflammation has been postulated as the cause of synaptic dysfunction and intestinal motility abnormalities at the early stage of PD progression since these EGCs regulate motility, intestinal permeability, and immune responses . This asIn addition, the "pathological loop" between glial activated by aggregated α-syn and the misfolded α-syn generated by intestinal glial activation emphasizes the significance of EGCs in the pathogenesis of PD . This reTaken together, these clinical and preclinical findings from patients and various animal models suggest that increased intestinal permeability, altered intestinal microbiota, intestinal inflammation, and EGC activation are early events of the disease that occur prior to the onset of CNS symptoms . At an eAs the population ages, the morbidity and mortality of PD are increasing year by year globally. Oral levodopa or a dopamine agonist is still the first-line treatment for patients with PD. Intestinal dysfunction may not only be the origin of PD but also affect the absorption of drugs in the gut, accelerating the progression of the PD. Current knowledge may aid in the development of novel therapeutic interventions targeting gut functions in the early stages of the disease. Developing therapies to minimize gut dysfunction might potentially slow or halt neurodegeneration ascending to the CNS. While more studies are needed to validate the exact role of enteric pathologic in the onset of PD. Fortunately, the development of experimental models enables us to gain a better understanding of the causality and relationship between intestinal dysbiosis, permeability changes, EGCs and intestinal inflammation in PD. Meanwhile, prospective longitudinal studies in subjects at risk for PD are needed to identify which factors act as triggers to cause or promote the disease. Changes in intestinal function may become a sensitive marker for predicting and assessing the risk of PD in the future.


Engineered Cementitious Composite (ECC) is currently receiving more and more attention due to its excellent tensile strain hardening and multiple cracking properties. However, due to the high material cost of polyvinyl alcohol (PVA) fiber and quartz sand, its widespread promotion and application in the market are limited. Therefore, scholars at home and abroad have conducted many active studies on improving ECC. This paper summarizes the development history and research status of ECC materials, summarizes the current domestic and foreign researchers’ improvement methods for ECC materials, and classifies the improvement methods into three categories: the type of fiber variation, the water-binder ratio variation and adding mineral admixtures, the influences of the above three factors on the mechanical properties and durability of ECC, such as compressive and flexural resistance, are described in detail, and the mechanism of action is explained. Furthermore, this paper introduces the most common uniaxial compression and uniaxial tension constitutive models of ECC. They are briefly classified and evaluated, hoping to help readers’ numerical simulation analysis. Finally, some suggestions for ECC research, such as the proportion of water binders and the application of composite fibers, require further research. Concrete has gradually become an irreplaceable building material in civil engineering due to its abundant access to raw materials, strong plasticity in engineering, good working performance, ability to adapt to different uses and environments, durability, and other advantages . With thWith the rapid development of urbanization and industrialization, the world population has grown exponentially . The earIn recent years, as the performance of FRC has shown strong excellence in the construction field, scholars at home and abroad began to modify and optimize them to obtain the characteristics of low cost and high strength to better and more widely benefit social undertakings. Currently, the modification of fiber-reinforced concrete can be divided into three categories: ① By changing the types of fiber mixed into engineering cementitious composites to change the characteristics. The current standard method is to mix polyvinyl alcohol fiber (PVA fiber), polypropylene fiber (PP fiber), carbon fiber (CF fiber), basalt fiber (BF fiber), and so on in concrete to improve the strength and crack resistance. ② The use of mineral admixtures instead of cement in engineering cementitious composites to improve performance. The current standard method is to use fly ash (FA), silica fume (SF), slag, and recycled powder (RP) to replace cement. ③ The fiber-reinforced concrete was modified with a coupling agent and other chemical substances.Engineered Cementitious Composites (ECC) are one of the FRC materials proposed by Victor in 1992 . Li usedHowever, despite its excellent mechanical properties and good durability, ECC removes coarse aggregate during preparation, increasing the amount of fine aggregate and cementitious material at a high cost. The most commonly used cemented material in traditional ECC is Portland cement. It will consume not only much limestone, sand, clay, and other natural resources but also have high energy consumption, significantly impacting the environment. The total emission of 5–7%  will aggIn order to reduce the cost of ECC and increase its green environmental performance, scholars have conducted much research on improving ECC. They change fiber types or add mineral admixtures to reduce manufacturing costs and increase their green environmental performance. Cui  replacedThis paper has three novel points compared to the existing literature. Firstly, the admixtures of engineering fiber cementitious composites studied in this paper are expanded, which studies the properties of composites mixed with different fibers and mineral admixtures. This paper mainly analyzes two categories: using various mineral admixtures to replace cement in ECC; the recycled micro powder used to replace quartz sand in ECC. There are few studies on recycled micro powder and engineering fiber cementitious composite materials in the current open literature. Therefore, this paper further studies the composite materials mixed with recycled brick and concrete powder. Secondly, when analyzing the fiber types and content of engineering fiber cementitious composite materials, there are two characteristic analysis methods in this paper, for example, the transverse comparison between the compressive strength of the same fiber under different dosages and the compressive strength of different fiber under the exact dosage. Most of the existing literature research data focuses on one fiber, and this paper is innovative in providing a deeper understanding of engineering fiber cementitious composites. The third innovation point is the detailed study of the constitutive structure of ECC, such as the uniaxial compression constitutive model considering dimension and linear and nonlinear analysis models. The three innovative points will better show the performance and characteristics of engineering fiber cementitious composites so that readers will understand them more comprehensively.This paper describes the research status of ECC materials, introduces the design theory of ECC materials in detail, summarizes the effects of fiber types, water-binder ratio, and mineral admixture types on the mechanical properties and durability of ECC materials, and elaborates on the mechanism of action in detail. Furthermore, the constitutive models of ECC uniaxial compression or tension are summarized and briefly evaluated. Finally, some suggestions on the future development direction of ECC are presented.The compressive strength of cement base usually uses the cubic compressive strength of China GB 50010-2010 “Code for Design of Concrete structure” as the fundamental strength index of concrete, in the structural design is used as the prismatic axial compressive strength of concrete design index of compressive strength. Because the uniaxial compression test is relatively simple and accurate, most domestic and foreign scholars use the uniaxial compression test to study the compressive strength of ECC.Fiber plays an essential role in high-performance concrete, and fiber incorporation can improve the mechanical properties of concrete by changing the cracking form . TherefoThe influence of fiber type on ECC performance mainly depends on the characteristics of the fiber itself. The polymerization of polyvinyl alcohol forms PVA fiber. Since the hydroxyl group of vinyl alcohol monomer is retained in the PVA molecule, the hydrophilicity of PVA fiber is good, which makes it have better dispersion than other fibers in the water-containing concrete mix . WhetherPP fiber is a low elastic modulus synthetic fiber with high strength, good ductility, excellent durability, and low price. Its raw material, the monomer C3H6, is a high molecular hydrocarbon, its chemical stability is good, and most chemicals do not react but has flammability, resulting in the reduction of the fire resistance of concrete. CF fiber is an organic fiber containing carbon. A graphite-like structure is formed in an inert atmosphere through multiple processes, such as pre-oxidation and high-temperature carbonization . As a reKuang  made sevScholars have made many achievements in the research on the compressive strength of different fibers mixed with ECC: Hao et al.  conducteThe influence of different fibers on the compressive strength of ECC is different. Domestic and foreign scholars mainly use PVA fibers to conduct experiments on the performance of ECC. Therefore, PVA fiber-reinforced cement base is discussed without special instructions. The water-binder ratio is an essential parameter of the concrete mix ratio, and the mechanical properties of concrete are directly related to the water-binder ratio. Under the condition that the variety, quality, and dosage of mixed materials remain unchanged, the water-binder ratio directly determines the strength of concrete. Therefore, scholars conducted a uniaxial compression test by changing the water-binder ratio to study the influence of the water-binder ratio on compressive strength. In the ECC compression test, Wang et al.  found thHowever, Tian  adopted It can be observed from ECC matrix materials are mainly made of cement, quartz sand, auxiliary cementitious materials, and fiber as reinforcement materials and are added into the admixture to achieve ultra-high toughness. Many researchers have improved ECC in recent years and designed cement-based composites with excellent properties for economic or environmental protection. It is divided into two categories: ① Various mineral admixtures are used to replace cement in ECC raw materials; ② Recycled micro powder is used to replace quartz sand in ECC material.2 and other substances. A large number of active ingredients SiO2 and Al2O3 in the fly ash will have a secondary hydration reaction with Ca (OH)2 to produce calcium aluminate hydrate and other substances, which fill in the gap of cement hydration products, enhancing the compactness of ECC and thus improving its strength. When the fly ash content is significant, the incorporation of fly ash reduces the cement clinker in the whole material, and the degree of hydration reaction of fly ash is lower than that of cement, so it cannot play the active certification role. Therefore, high fly ash content will reduce the strength of ECC, and the higher the fly ash content, the more noticeable this phenomenon is. Turk et al. [As for the use of mineral admixtures to replace cement in ECC raw materials, the mineral admixtures used by domestic and foreign researchers mainly focus on fly ash, silica fume, and various solid waste powders. When fly ash replaces cement with different contents, the conclusions on its influence on ECC compressive strength are consistent. Wang , Tan et k et al.  studied Silica fume replaces cement in ECC with different substitution rates, and its influence on compressive strength is consistent with fly ash replacing cement. See As for replacing quartz sand in ECC material with recycled micro powder, domestic and foreign researchers mainly focus on two kinds: recycled brick powder and recycled concrete powder. Li et al.  studied The advantage of ECC material performance is mainly reflected in its tensile performance, with prominent stress-strain characteristics. Because the uniaxial tensile experiment is simple to operate, has low requirements on test instruments, and can effectively reduce the influence of eccentric tension on test results, high success rate, and accurate test results. The uniaxial tensile tests are used to evaluate the unique mechanical properties of ECC when studying tensile strength.Nowadays, the tensile strength of PVA fiber, P.P. fiber, basalt fiber, and carbon fiber mixed with ECC is mainly studied at home and abroad. Different types of fiber have different tensile properties. Yao et al.  tested tStudies on factors influencing the tensile properties of ECC by domestic and foreign scholars mainly focus on the experiments of PVA fiber. Therefore, studies are conducted on PVA fiber-reinforced cement base without particular explanation below. The PVA dosage will directly affect ECC’s tensile strength, and studying the optimal dosage helps reduce the cost and enhance performance. Luo et al.  found thTheoretically, the increase in the water-cement ratio will reduce the strength of the cement base and, thus, the tensile strength of PVA-ECC . HoweverBased on the research of many scholars, the relationship between the change of effluent glue ratio, ultimate tensile strength, and ultimate tensile strain can be obtained, as shown in In recent years, Li et al.  have beeGenerally, when studying the compressive strength of engineering fiber cementitious composite materials, scholars adopt the uniaxial compressive test method, and when studying tensile strength, the uniaxial tensile test is adopted. They all establish linear models through fitting to conclude mechanical properties. However, the final applications are different due to the variety of composite materials. For the engineering fiber cementitious composite mixed with different fibers, although the tensile strength of PVA fiber is not as high as PE fiber, its price is much lower than PE fiber on the premise that it can meet the requirements of ECC design theory on fiber. Therefore, in recent years, PVA fiber has gradually replaced PE fiber. More and more people are studying PVA-ECC in China. Another example is a mixed fiber-reinforced cement-based composite material. It can make up for the shortcomings of a single fiber to have better all-around performance. Under the premise of meeting the requirements of use, we can consider the fiber cost, environmental protection, and other factors. Therefore, composite materials can be more practical, and scholars focus on mixed fiber cement composites.Mineral admixtures include fly ash, silica fume, and a variety of solid waste powders. Fly ash and silica fume have the same characteristics when they substitute cement into ECC. Therefore, increasing the substitution rate has positive effects within a specific range. Considering the compressive strength of ECC, the dosage of mineral admixture instead of cement in ECC should be a manageable size .Therefore, the difference in composite materials gives the application range its characteristics. In practical life, the application of composite materials should be determined according to the specific practical needs.ECC has prominent strain-hardening properties under bending loads . In receThe flexural strength of different fiber types will change after the addition of ECC. Scholars at home and abroad have mixed different kinds of fiber into ECC and measured its flexural strength by a four-point bending experiment. Liu et al.  found thGenerally, the minimum fiber volume to maintain strain hardening is called the critical volume. The ECC cannot have good bending strength if the fiber content is lower than the critical volume. Due to the high price of fiber, it is feasible to reduce fiber content at the expense of ECC toughness to a certain extent in some specific situations. Zheng et al.  carried According to the four-point bending experimental data, Du  found thFly ash not only affects the compressive strength of ECC material but also significantly affects the bending toughness of ECC material. Chen  set the Drying shrinkage is an inherent property of cement-based materials. In a dry environment, the internal humidity of cement-based materials is greater than the ambient humidity, resulting in water loss in the hydration phase. In an engineering environment where the relative humidity is greater than 40%, the capillary force and dismounting pressure caused by water loss cause the cement-based materials to shrink, and the drying shrinkage accounts for 80–90% of the total shrinkage . Its 28-The water-binder ratio is the proportion of each material in PVA-ECC, which affects the amount of hydration reaction and causes altered chemical contraction and self-contraction. For different water-binder ratios, the drying shrinkage of ECC increases with the water-binder ratio, and the smaller the water-binder ratio is, the more pronounced the increase is . It is bThe fiber content affects the mechanical properties of ECC and has a subtle effect on its drying shrinkage. Meng et al. , a forei−6, lower than or slightly higher than the benchmark concrete. Lee et al. [As a kind of volcanic ash mineral admixture, fly ash can effectively improve the durability of cement-based materials, and different fly ash admixtures have specific effects on the drying shrinkage of the cement base. Domestic and foreign scholars have conducted dry shrinkage experiments on ECC specimens with different dosages, but the experimental results are slightly different. Chen et al.  in Chinae et al.  believede et al.  researchIt is necessary to study the basic mechanical properties of ECC material level intensely if ECC material is used in building structures. Therefore, establishing the ECC constitutive model is crucial for studying ECC components and even the structural level. Therefore, the two typical ECC constitutive were introduced: tension constitutive and compression constitutive.Due to the brittleness of concrete material, it is mainly used to withstand pressure in practical engineering. According to the ECC dense accumulation principle, the usual coarse aggregate in traditional concrete is removed, which makes the matrix structure dense and has higher compressive performance and durability. Therefore, it is necessary to explore the law of ECC’s compressive performance . The uniThrough uniaxial compression tests, domestic and foreign researchers generally obtain the dimensionless stress-strain curve, as shown in the α = cf/cuf. Qin [Researchers at home and abroad have simplified stress-strain curves to varying degrees and established corresponding constitutive models, which can be divided into linear and nonlinear models. Zhu et al.  built a fcu. Qin  has the fcu. Qin  directlyfcu. Qin  team conFor the nonlinear analysis model, Guo  proposedThe tensile strength essentially controls the mechanical behavior of structural members. In practical engineering, the calculation of the bearing capacity of structural members, such as shear, torsion, and cracking, is related to the tensile strength of concrete . The excScholars at home and abroad analyzed the mechanical properties of ECC, drew the stress-strain curves through uniaxial tensile experiments, and established various uniaxial compression constitutive models. The most representative is foreign scholar Kanda et al. , who proAfter ignoring the expression of the strain-softening segment, the constitutive equation for the first two stages is:n et al.  took theXu et al.  proposedWhen the specimen is at the starting point B of the rear multi-crack development section, the stress transmitted by the fiber at the crack interface cannot cause the matrix to generate new cracks. It is because the bearing capacity of the cement matrix has been completely lost. The stress is all borne by the fiber. With the increase of strain, the fibers at the crack interface are gradually pulled off or pulled out from the matrix. Due to the slip-hardening effect of the fibers, the stress of ECC material gradually increases until the material’s tensile strength is reached, the curve reaches peak point C, and the multi-crack opening section ends. Therefore, the expression of the three-fold stress-strain curve of Xu  is as foThe constitutive mechanical structures of engineering fiber cementitious composites have been summarized, and their characteristics, advantages, and disadvantages are included in Under normal conditions, the structure layout is reasonable if the concrete structure design is proper and the construction quality is reliable. As a result, the structure has good working performance. However, when the structure suffers from various external uncontrollable factors such as accidental load, structural function change, deterioration of service conditions, or improper maintenance, the applicability, durability, and safety of the concrete structure will be affected. Therefore, studying ECC’s durability for future ECC applications is essential.One of the most dangerous environmental conditions for concrete is the freeze-thaw cycle. In the cold area of north China, the freeze-thaw cycle is often the main factor leading to the deterioration and destruction of building structures. Fatigue stress quickly occurs in the building structure damaged by the freeze-thaw cycle, which leads to internal damage and strength reduction. If it acts on the pavement, it will cause surface erosion and many cracks. In the bridge, it will cause cracking of the concrete surface layer, denudation of mortar, and exposure of coarse aggregate, as well as exposure and corrosion of reinforcement . To solvCracks are inevitable in the concrete structure; water, corrosive substances, and oxygen will enter the concrete through cracks and corrosion, thus reducing the structure’s service life. Lepech et al.  studied ECC structure is subjected to two types of fatigue loads: one is dynamic effects such as vehicles, earthquakes, wind, and waves; the other is due to the dramatic changes in the external environment, which makes the structure experience temperature difference and freeze-thaw repeatedly, thus generating indirect cyclic stress inside . Under rChai et al.  conducte(1)With the increase in fiber content, the compressive strength of the PVA-ECC cube and the axial compressive strength are both improved. When the fiber content is between 1.5% and 2.5%, the overall compressive strength of the cement-based material will be increased. However, the compressive strength will decrease significantly when the fiber content exceeds 2.5%.(2)The increased water-binder ratio decreases compressive strength gradually. As a result, the relative proportion of cement becomes smaller, and it cannot fill the pores left by sand and stone aggregate, resulting in more significant porosity and gradually reduced compressive strength.(3)Within a specific range, the replacement of cement by fly ash has a positive effect on the compressive strength of ECC, which can be improved by about 20–30%. However, if the content exceeds a specific limit value, the compressive strength of ECC will decrease rapidly as the replacement rate of fly ash continues to increase.Uniaxial compression characteristics of PVA-ECC(1)The cracking strength, cracking strain, ultimate tensile strength, and ultimate tensile strain of the ECC thin plate increase with the increase of PVA fiber volume content. For example, when PVA fiber volume content is 2.0%, the bridging effect of fiber can be fully exerted, and the tensile performance of ECC is also the most significant improvement.(2)With the increase of the water-binder ratio, the ultimate tensile strength of PVA-ECC decreases at 7 and 28 d, but the tensile strain hardening characteristics and ductility of PVA-ECC are obviously improved. Therefore, the water-binder ratio should be selected when preparing PVA-ECC.(3)When mineral admixture is added to ECC, the ultimate tensile strength decreases, but the ultimate tensile strain increases with the admixture amount.Uniaxial tensile characteristics of PVA-ECC(1)With the increase of PVA fiber volume content (1.0%→2.0%), the toughness index of ECC firstly increases and then decreases and reaches the maximum at 1.5%. Moreover, increasing PVA fiber content can improve the initial crack strength and strain hardening effect when the first crack appears.(2)With the increase of fly ash content, the crack width and free drying shrinkage of the ECC structure decrease significantly. As a result, the interfacial bonding force between PVA fiber will be improved effectively.Bending strength of PVA-ECCCompared with conventional concrete, ECC has strong freezing resistance, permeability resistance, and anti-fatigue performance, which makes ECC have a broad application market and occupy a place in nowadays bridge construction, pavement laying, and other aspects.In this paper, the development prospect of engineering fiber cementitious composites is first introduced. Then the basic mechanical properties and durability of engineering fiber cementitious composites are studied by uniaxial compression, uniaxial tensile, and conventional bending tests, respectively, according to fiber types, mineral admixture types, water-binder ratio, and other factors. According to the stress-strain curve, various constitutive equations suitable for PVA-ECC are proposed. Through the above research, the main conclusions are as follows:This paper summarizes the mechanical properties of ECC, summarizes the conclusions from three aspects: fiber type, water-cement ratio, and mineral admixture, respectively, in terms of compressive resistance, tensile resistance, and bending resistance, and classifies and summarizes the research results of domestic and foreign scholars. It is found that the current research on ECC needs to be improved in the following aspects, and further research can be conducted from these aspects. Furthermore, to better develop the scope of future ECC applications.(1)At present, scholars at home and abroad focus on studying the mechanical properties and durability of PVA-ECC. However, there is still a need for more exploration of other fibers, such as basalt fiber and carbon fiber, there still needs to be more systematic and comprehensive research, and the research level only stays on the surface. Therefore, it is expected to find cheaper and better-performance fibers to replace PVA fiber in the market by increasing the research on other fibers.(2)More experiments must be conducted on mixing hybrid fibers into a cement base. Currently, PVA-steel fibers are often mixed for experiments at home and abroad, and the data needs to be more comprehensive. The subsequent research on single fiber can be expanded to study hybrid fibers.(3)The water-binder ratio of ECC is complex in mechanical research. Domestic and foreign scholars have reached conclusions by changing the water-binder ratio in mechanical tests, but opposite conclusions have occurred many times. Therefore, researchers should continue to study the theoretical and practical points of the influence of the water-binder ratio on mechanical properties in the future to form a set of confirmed water-binder ratio theories.(4)To replace cement with mineral admixtures, fly ash, and silica fume have been fully studied. Subsequently, studies can be conducted on waste ceramic powder and glass powder to enhance the utilization of solid waste materials. Furthermore, reclaimed micro powder is an emerging research project nowadays. For example, the reclaimed brick powder studied in Tian  can not


Pressure ulcer (PU) and burn injury (BI) represent two types of wounds that require broad and difficult fields of treatments. Despite various advances made in recent years, these injuries have few solutions that allow recovery in shorter times and with greater effectiveness. All this negatively affects the patient's quality of life. Since ancient times, with the use of torpedoes (a kind of fish capable of producing electric discharges), it has been believed that the use of electricity could favor the repair processes of various kinds of wounds.Today, technological evolution has allowed the creation of more and more advanced techniques that can determine a better reparative response of the injured tissues. The radio electric asymmetric conveyer (REAC) technology is one of these and the reparative tissue optimization (TO-RPR) treatment represents the specific treatment for these lesions. The two cases presented in this article are intended to highlight how two serious injuries of a different nature, when treated with the REAC TO-RPR, have the same rapid qualitative and quantitative recovery path that continues even after the end of the treatment cycle. The stability and progression of the effects are typical of REAC treatments, and in this article, it is possible to appreciate the clinical evidence. These results together with others previously published open a new therapeutic possibility in the treatment of wounds. A wound is generally considered to be a rupture in the integument of an organism produced by acute trauma or chronic processes, which include but are not limited to the following etiologies: venous ulcers, diabetic ulcers, and pressure ulcers (PUs), involving signs that trigger a multitude of biological phenomena aimed at restoring the functional integrity of tissues .The wound itself is a sign of damage and, in still viable organisms, the resulting biological mobilization is aimed at repair, starting from an instantaneous physical event that is often overlooked, but that accompanies every wound, a dramatic change in the electrophysiological character of the intact skin surrounding the wound .PU, a debilitating chronic wound, is a significant and still common problem in all health settings, affecting approximately 10% of hospitalized patients, especially in intensive care units ,4.PU is a usual problem especially in elderly patients with immobility and inactivity due to significant co-morbidities such as diabetes and arterial vascular disease, carrying very significant human and economic impacts ,6.PUs represent a major burden to patients, including negative psychological, physical, and social consequences affecting health, well-being, and health-related quality of life . EffectiBurn injury (BI) represents another interesting chapter of vulnology . BIs, ifSome radio electric asymmetric conveyer (REAC) technology treatments have demonstrated reparative and regenerative efficacy -13. AmonThe REAC technology was designed for neurobiological stimulation treatments through the reorganization of the endogenous bioelectric activity at the molecular level, according to precise therapeutic protocols, for the various indications of use.The therapeutic effect is determined by the interaction of the radio electric field generated by the REAC device with the molecular structures of the tissues through the ACP connected to the device. This interaction optimizes the endogenous bioelectric activity. The effects are diversified according to the type of treatment protocol used. The TO-RPR type has been designed to promote reparative processes.REAC TO-RPR treatments are treatments preprogramed for their intended use by the manufacturer of the REAC medical devices and cannot be changed in any way by the operator. There are no contraindications with the use of drugs of various nature and indications.The REAC TO-RPR in these two reported cases was administered twice a day, for a total of 18 treatments of 15 minutes each.The REAC model device used in this study was BENE 110 .Case 1The first case concerns a patient suffering from a PU. The patient is a 70-year-old Caucasian man, with a history of hypertension and diabetes for 12 years, having undergone angioplasty six years ago, presented with ischemic stroke with hemorrhagic transformation on January 16, 2020. Being submitted to decompression craniotomy plus duroplasty, he remained hospitalized for a period of two months, during which time he developed an extensive PU in the sacral region, despite the fact that he was on an anti-bedsore mattress.At our initial examination, on March 12, he presented a delimited, deep lesion, full thickness tissue loss with exposed muscle, erythematous borders, a bed filled with sloughs, moderate serous exudation, without odor, hemorrhage, and signs of infection, NPUAP/EPUAP Pressure injury classification Stage IV . In addition to the short healing time, a new hydrated tissue, without retraction stands out. The recovery of the tissue's physiological pattern and the refined reparative process are highlighted by the regrowth of skin appendages such as hair also on the scar surface, which is not observed in processes of this nature in conventional treatments . The extended BI was provoked by an act of self-harm. The subject in question set fire to the surface of a soup spoon and once it became incandescent, using its convex part, it began to produce lesions. The patient repeated this self-injurious act several times to cover the entire surface of the left forearm Figure .Initially attended at the reference service Burn Treatment Center on June 14 after preliminary evaluation, he underwent a surgical procedure for tangential debridement to remove devitalized tissue. On the same day, he underwent a new tangential debridement for the removal of devitalized tissue Figure .Since the patient had already been involved in similar acts several times, the doctors decided not to proceed with a skin graft transplant, and the patient was referred to our service. The patient's situation at the time of our first observation on 22 June is shown in Figure In this case, we used the same cycle of 18 sessions of REAC TO-RPR treatment, each lasting 15 minutes. The positive and rapid evolution of the reparative processes of BI can be observed in Figures Since pain represents an important component in this type of injury, especially during handling for cleaning and daily dressings, we have decided to evaluate the perception and communication of pain using a visual analog scale, to be administered at each session of REAC TO-RPR treatment Figure .The perception and communication of pain trend during the REAC TO-RPR treatments are summarized in Table The results reported in Table Regenerative medicine has always focused its attention fundamentally on chemical-biological tools, probably because they are potentially more easily produced and used on a large scale. All this probably had a negative impact on the promotion of the search for the therapeutic manipulation of endogenous ion flows (EIF). EIFs play a key role in regulating cellular behavior by imperatively conditioning the processes of cell repair and regeneration .Repair and regeneration of injured or diseased tissues are complex biological processes, during which many molecular, cellular, and tissue responses are involved , such asThe initial inflammatory response leads to the influx of macrophages and neutrophils, which release cytokines, growth factors, and nitric oxide, and induce nearby keratinocytes to migrate across the wounded epithelium .While cytokines and growth factors can indeed stimulate keratinocyte migration, a much earlier stimulus of directed migration is triggered by wounding: the electrical field generated by the flow of current out of the wound .Endogenous bioelectric fields (EBFs), detected in wounds and damaged tissues, can control cell differentiation, proliferation, and migration, enabling the robust restoration of the normal pattern after injury . The recDifferent treatment strategies exist for the management of chronic wounds: some are invasive, such as wound debridement and skin substitute therapy; while others are noninvasive, such as compression bandaging, wound dressing, hyperbaric oxygen therapy, negative pressure therapy, ultrasound, and electrostimulation therapy (EST) . The REAA clinical demonstration of this claim is represented by the cases described in this article.Although the two lesions described have different origins, the manipulation of the endogenous bioelectric activity appeared to have led the two lesions to a common healing process that has proven to be rapid and refined compared to the classic expectations of healing with respect to these lesions.The REAC TO-RPR and regenerative tissue optimization (TO-RGN) treatments represent a powerful tool for neurobiological modulation, able to modulate, each for its own specific indications, the bioelectrical signals that control cell proliferation, migration, and differentiation. For this mechanism of action, they can affect the treatment of difficult wounds speeding up healing times, regardless of the cause that induced the injury. They favor a refined restoration of the damaged tissue, to allow the reappearance of the skin appendages also on the scarred area. Moreover, the speed and quality of the reparative process allow a rapid disappearance of pain in the BI case. Further studies can be useful to consolidate these results.