Real‐Time Monitoring of Wound States via Rationally Engineered Biosensors

Current strategies for real‐time wound monitoring have limited applicability and therapeutic efficiency due to the reliance on clinician experience and time‐consuming laboratory analysis requirements. In this article, an educational review is provided on the accumulated knowledge of wound healing, such as the characterization of acute and chronic wounds, the four main stages of wound healing, and the factors that influence wound healing. The current state‐of‐the‐art wound state sensing methods are presented and reviewed, with particular attention paid to the real‐time monitoring of pH, biofilms, electrophysiological signals, temperature, and reactive oxygen species. It is expected that the presentation of this evolving field will contribute to deepen the existing understanding of wounds and steer wound treatment toward real‐time precision to drive further and faster development of clinically valuable technologies.


Introduction
The skin is the largest organ of the human body, mainly responsible for protecting the human body from external damage, regulating body temperature, and sensing external stimuli. [1]The rupture or defect of normal skin under the action of external injury factors is called wound, which is often accompanied by the destruction of skin integrity and the loss of a certain amount of normal tissue. [2]Inefficient treatment of wounds increases the risk of exacerbations such as bacterial infection, severe bleeding, and long-term unhealing.Efficient wound care has been a major and costly medical challenge on a global scale, with important socioeconomic implications at scales ranging from individual to healthcare and policy-maker levels. [3]ccording to the state and duration of the healing process, wounds can accordingly be classified as either acute or chronic. [4]OI: 10.1002/adsr.202200018   The process of wound healing involves four overlapping and sequential stages, including hemostasis, inflammation, proliferation, and dermal remodeling. [5]any factors can affect one or more of the four stages of wound healing.Interruption, abnormality, or prolongation of each stage can delay wound healing or turn it into a chronic, non-healing wound. [6]Familiarity with these objective laws of wounds is a prerequisite for the development of advanced dressings and the adoption of the correct treatment.To the best of our knowledge, there are currently only a few systematic summaries and analysis of wound healing, which seriously hinders the improvement of related technologies and nursing methods.In addition to analyzing and summarizing the objective laws of wound healing, it is also of great significance to perceive the wound state in real time and guide doctors to take correct treatment methods. [7]For example, pH and temperature at the wound site can serve as indicators of bacterial infection and inflammation, while also influence a range of chemical and enzymatic reactions during wound repair. [8]Moreover, the formation of wound biofilms hinders the healing of 6% of acute wounds and 60% of chronic wounds and reduces bactericidal efficiency. [9]Meanwhile, the physiological state of the wounded has a non-negligible impact on wound healing, and it has also become an entry point for doctors to analyze and diagnose pathological information. [10]owever, most wound treatments in the clinic are mainly based on visual observation, or rely on complex and time-consuming biochemical analysis to sense the state of the wound. [11]These methods can easily lead to misdiagnosis and delay the timely treatment of the wounded.
In this review, we highlight the healing analysis and real-time sensing wound status, aiming to deepen the existing understanding of wounds and guide wound treatment toward real-time precision.First, acute and chronic wounds are described in detail regarding their characteristics and causes.Afterward, the four main stages of wound healing are introduced in detail, and the changes in each stage are analyzed for both their macroscopic performance and their microscopic mechanisms (changes at the tissue, cellular and molecular levels).Furthermore, the most significant factors (infection, ROS, diabetes, and age) that affect wound healing are discussed.Finally, we elaborate on the current state-of-the-art wound state sensing methods, with a particular attention to the real-time monitoring of pH, biofilms, electrophysiological signal, and temperature.

Classification of Wound
Wounds have many causes, namely war, surgery, burns, or pathologic conditions (such as vascular diseases or diabetes).According to the state and duration of the healing process, wounds can accordingly be classified as either acute or chronic. [4]1.1.Acute Wounds Scalds, burns, abrasions, and surgical incisions are all acute wounds, which have the characteristics of a short healing time and normal inflammatory response following conventional healing procedures.Generally speaking, acute wounds are moist, with a small amount of wound exudate, no slough tissue, and no unusual smell.However, if an acute wound does not follow the correct healing pattern, it could become a chronic wound, delaying recovery.[12] At present, the difficulty regarding acute wound repair is to prevent scar formation.

Chronic Wounds
Chronic wounds, e.g.venous and arterial leg ulcers, diabetic foot ulcers, and pressure ulcers, have a non-healing or slow-healing tendency. [13]The common characteristics of these wounds include long-term inflammation, persistent infection, microbial biofilm formation, and the inability of the appropriate cells to respond to repair stimuli, which together prevent the wound from healing properly. [14]Unlike acute wounds, the healing of chronic wounds stagnate in the inflammatory and proliferation phases, [15] and this failure to heal is currently the most difficult medical problem.The causes of chronic wounds are complex, mainly including venous insufficiency, peripheral vascular diseases, systemic diseases, traumatic scars, and infections.The factors affecting wound healing are also complex and diverse, including systemic and local factors.Chronic diseases, vascular problems, diabetes, neuropathy, malnutrition, advanced age, stress, infection, edema, etc., all of which can hinder wound healing.Moreover, an increase of senescent cells, imbalance of enzymes, and decline in the availability of growth factors also exacerbate the state of chronic wounds. [16]aking arterial ulcers as an example, it is caused by insufficient blood supply to the skin, of which atherosclerosis is the main cause. [17]Typical arterial ulcers often occur on the extremities, such as toes, heels, and other protruding parts of the foot.The main symptoms are intermittent claudication and rest pain.Ulcers are usually well-circumscribed, pale wounds, thickened toenails, gangrene of the extremities, etc. [18] Common treatments include infection control, improved circulation, wound protection, and nutritional support.

Wound Healing Stages
Wound healing is a highly conserved and dynamic process involving complex and coordinated interactions of multiple cell types and growth factors.These factors drive the overlapping phases of wound healing: hemostasis, inflammation, proliferation, and dermal remodeling (Figure 1). [5,19]In this section, the four main stages of wound healing are introduced in detail, and the changes in each stage are analyzed for both their macroscopic performance and microscopic mechanisms (changes at the tissue, cellular and molecular levels).These will greatly deepen our understanding of the mechanisms involved in wound repair and provide ideas and directions for the design and implementation of future dressings.

Hemostasis
Three steps occur in physiological hemostasis, and these include vasoconstriction, platelet thrombosis and blood coagulation.Immediately after injury, local vascular smooth muscle cells contract to reduce blood loss.This is caused by vasoconstrictor agents, such as endothelin, which are released from damaged endothelial cells. [20]In addition, platelet-derived growth factor (PDGF) produced by platelets activates mesenchymal cells, notably smooth muscles of blood vessel walls that cause contraction. [21]However, initial vasoconstriction is limited and only temporarily reduces bleeding.In the second stage, platelets adhere strongly to the exposed collagen fibers of damaged blood vessels, and cluster together to form hemostatic thrombi.A thrombus directly blocks the opening of the blood vessel, acting as a plug, but it also maintains the integrity of the blood vessel wall.After a blood vessel is damaged, the coagulation factor is activated to generate thrombin, which converts the soluble fibrinogen in plasma into insoluble reticulofibrin, thereby enhancing hemostasis. [22]The hemostatic fibrin clot formed by platelets and coagulation cascade factors also has many secondary functions, e.g., preventing bacterial invasion, providing a scaffold for immune cells and a repository of cytokines and growth factors that guide the early repair of the damaged cells.

Inflammation
Congenital inflammation has evolved as the main defense against pathogenic microorganisms that invade wounds.This immune response starts within a few hours of injury, fueled by the products of bacterial degradation, the degranulation of platelets, and the activation of complement. [19]Neutrophils migrate from the circulating blood into the infected wound site after injury, degrading damaged matrix proteins and killing bacteria. [23]Resting neutrophils are round cells with some membrane folds that form prominent extracellular structures and fibers after being stimulated with phorbol myristate acetate, interleukin-8, or lipopolysaccharide, by which they become activated.Even before bacteria begin to be engulfed by neutrophils, these cells produce extracellular fibers, degrade damaged matrix proteins and kill bacteria.Furthermore, the extracellular fibers can act as a physical barrier that keeps damaged matrix proteins and bacteria from diffusing away, preventing damage to tissues adjacent to the site of inflammation. [24]Circulating monocytes enter the wound tissue within 24 h, differentiating into macrophages that remove tissue debris, destroy bacteria, Figure 1.The four stages, key to molecular and cellular of wound repair: A) hemostasis, B) inflammation, C) proliferation, and D) remodeling.Reproduced with permission. [5]Copyright 2014, American Association for the Advancement of Science.
facilitate the non-phlogistic removal of neutrophils and prepare for angiogenesis and tissue granulation formation. [25]

Proliferation
As the inflammation subsides, wound repair enters its third stage, that of proliferation.This phase can be divided into three steps: re-epithelialization, restoration of vascular network, and formation of granulation tissue, which are mainly the results of cellular proliferation and the migration of many cell types. [26]Under the influence of several growth factors, including transforming growth factor-, epidermal growth factor, and keratinocyte growth factor (KGF), keratinocytes start to proliferate and migrate, thus skin wounds re-epithelialize. [27]The leading edge of these skin epithelial cells is pulled forward by the contraction of actin cables that close the gap (Figure 2A). [28]As an epithelial wound repair, the front row of cells changes their shape and retreats into the back row (e.g., cells marked in yellow), un-til the wound eventually closes like a pinhole (Figure 2B). [29]ctin-rich filopodia (blue mark) from 2 opposing epithelial cells on membrane contact and subsequently produce tight junctions through adhesion (Figure 2C).Filopodia can be used as "sensors" of frontal epithelial cells to provide guidance cues and navigate to their targets.(Figure 2D and E). [30]The encounter and mature adherens junctions of filopodial interdigitation epithelial cells can be seen by transmission electron microscopy in Figure 2F.When epithelial cells contact and form new mature adhesion junctions, migration stops and they then secrete proteins to rebuild the basement membrane. [28]n essential activity in the proliferation stage is the formation of a new functional vascular network through angiogenesis. [31]n healthy tissues, the microvasculature system maintains a state of homeostasis for sufficient exchange of oxygen and nutrients, with the removal of carbon dioxide and waste products. [32]pon injury, destruction of the local vasculature and acceleration of metabolism at the injured site results in the wound tissues becoming hypoxic, which is a significant stimulus for Reproduced with permission. [28]Copyright 2001, Springer Nature.
angiogenesis. [33]This is mainly achieved by promoting the expression of vascular endothelial growth factor (VEGF).Low oxygen levels in wound tissues activate the hypoxia-inducible factor (transcription factor), which results in transcription of the VEGF gene. [34]In response to VEGF stimulation, endothelial cells break away from the basement membrane that normally binds them, allowing the cells to migrate, thus starting the formation of new blood vessels.First, angioblasts differentiate into endothelial cells, with subsequent vessel sprouting (angiogenesis) producing cords, developing lumens and remodeling into arterioles and venules. [35]Recruitment of vascular smooth muscle cells and pericytes that enwrap nascent endothelial cell tubules regulates perfusion and stability (Figure 3A). [36]Strictly speaking, the term "angiogenesis" mainly refers to sprouting from pre-existing blood vessels. [37]Attracted by proangiogenic signals, endothelial cells are activated and protrude filopodia.The tip cells are anastomosed with cells from adjacent sprouts and stalk cell rearrangement to build lumen.Finally, the lumen is extended, blood flows, and vessel maturation occurs (Figure 3B).The initiation of blood flow, occlusion and retraction of low flow vessels, pericyte coverage and branch regression leads to the maturity of newly formed blood vessels.The sprouting process is repeated until the proan-giogenic signal weakens and a normalized state is re-established (Figure 3C).
In the proliferation phase, a granulation tissue replaces the fibrin-/fibronectin-based temporary wound matrix, mainly composed of numerous fibroblasts and capillaries. [19,38]Its appearance is bright red, granular, soft, and moist, resembling tender granulation, hence the name of granulation tissue.Its microstructure is characterized by fibrocytes scattered in the middle of a capillary network and infiltrated by many inflammatory cells.The tissue contains some edema fluid, but there are no nerve fibers, making it impossible to perceive pain.Granulation tissue has the following essential roles in the process of tissue damage repair: 1) fighting infection and protecting wounds; 2) filling wounds and tissue defects; and 3) absorbing and replacing inactivated tissues or other foreign bodies.Granulation tissue can appear within 2 to 3 days after tissue injury, filling the wound and/or replacing foreign objects.In time, the granulation tissue gradually matures in order of its growth.The main morphological signs are: 1) water absorption decreasing gradually; 2) inflammatory cells decreasing and disappearing gradually; 3) capillaries becoming occluded and their number decreasing -a few capillaries are rebuilt into small arteries and venules; 4) collagen fibers Steps of vessel sprouting: sprouting, guidance, branching, anastomoses, and lumen formation.C) The process of vascular remodeling, from a primitive toward a stabilized and mature vascular plexus (from left to right).Reproduced with permission. [37]Copyright 2011, Cell Press.
produced by fibroblasts increase, becoming fibrocytes.Eventually, granulation tissue matures into fibrous connective tissue, transformed as scar tissue.

Remodeling
Remodeling is the last stage of wound repair, starting from 2-3 weeks after the injury, and lasting for one year or more. [19]t is characterized by decreased vasculature and cellularity, but with increasing tensile strength. [39]In this phase, type III collagen (the main component of granulation tissue) is actively remodeled into the stronger type I collagen as wound remodeling progresses. [40]This process is controlled by several matrix metalloproteinases secreted by macrophages, epidermal cells, endothelial cells, and fibroblasts, which can increase the strength of the repaired tissue. [41]The breaking strength (the tension at which the skin breaks) of wounded skin increases at this stage, regaining 70% of its initial strength after one year. [5]The newly created blood vessels at the proliferation stage are leaky, with a lack of tight junctions between cells, and they may also lack peri-cyte coverage.During remodeling in the further development of the cardiovascular system, well-perfused and stable mature blood vessels are gradually formed, which effectively reduce the leakage of substances. [42]In addition to the recovery of blood vessel function, the wound tissue can obtain sufficient oxygen, thereby stopping the production of VEGF and new blood vessels.Apoptosis of endothelial cells leads to the degeneration of any excessive blood vessels, and thus the bright red granulation tissue gradually recovers to a color similar to that of the surrounding skin. [43]

Factors Affecting Wound Healing
Many factors can affect one or more of the four stages of wound healing.Interruption, abnormality or prolongation of each stage can delay wound healing or turn it into a chronic, non-healing wound.Copyright 2021, Mary Ann Liebert Inc. B) Bottom cross-section image and color stereo image of growing Vibrio cholerae biofilm cluster at 18 h.C) Schematic diagram of the steps in Vibrio cholerae biofilm formation.The matrix is pink and the cells are yellow.D) Changes of verticalization time after using chemicals to control the cell length of Vibrio cholerae.Reproduced with permission. [52]Copyright 2019, Cell Press.E) Influence of ROS to modulate key events that occur during hemostasis, inflammation, proliferation, and dermal remodeling phases.Reproduced according to the terms of the CC BY license. [53]Copyright 2018, The Authors, published by MDPI.

Infection
Wound infection is a pathological response of microorganisms that invade the body through the wound, and that grow and multiply in the body, causing damage to normal bodily functions, metabolism, and tissue structure. [45]After the body is attacked by pathogens, it mobilizes defense functions to eliminate pathogens and their toxic products in order to restore the relative stability of the body.Whether a pathogen can cause infection after invading the body mainly depends on its virulence and the body's resistance to it. [46]Common pathogenic bacteria mostly include Staphylococcus aureus [47] Escherichia coli, [48] and Pseudomonas aeruginosa. [49]The typical symptoms of wound infection are local swelling, fever, suppuration, and a peculiar or foul smell (Figure 4A). [50]any bacteria that cause infection secrete polysaccharide matrices that form a biofilm adhering tightly to the wound.These biofilms reduce the efficiency of killing bacteria by external factors, e.g., immune cells and antibiotics.They are a key factor in the leading of wounds to be chronic, and create one of the most difficult problems in the treatment of wound infections. [51]Yan et al. [52] introduced the formation process and 3D structure of the Vibrio cholerae biofilms in detail.In a living, growing Vibrio cholerae biofilm, radial cells are located around the biofilm and vertical cells are located at its center (Figure 4B).When a single bacterium is present on the wound surface, it begins to replicate and expand outward to form a 2D single layer film.In this process, the bacteria in the center become under increasing pressure, which eventually exceeds the adhesion force between the bacteria.The central bacteria transform from a parallel base arrangement to a vertical arrangement.When vertical cells divide, they send out their offspring into three dimensions, so the biofilm transforms from a 2D surface to a mature 3D community (Figure 4C).After using chemicals to control the cell length of Vibrio cholerae, the verticalization time changes: the biofilm of longer (shorter) Vibrio cholerae transitions from 2D to 3D later (earlier) than Vibrio cholerae of normal length (Figure 4D).The experimental results are in line with the expected results, because shorter (longer) Vibrio cholerae require a lower (higher) critical force to promote verticalization.The result of changing Vibrio cholerae length is to change the aspect ratio of the biofilm they produce.

Reactive Oxygen Species (ROS)
ROS is a family of molecules with strong chemical reactions that mainly include hydroxyl radical (•OH), hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2 ), and superoxide anions (O 2 − ). [54]In wounds, the living activities of different cells and bacteria are constantly producing, transforming and consuming ROS.Mitochondria are the main production sites of ROS.In the process of oxidative phosphorylation, mitochondria oxidize organic fuel molecules to produce ATP, but at the same time they also produce ROS. [55]There is increasing evidence that ROS centrally affects all wound healing processes (Figure 4E). [53]At the same time, redox-based therapeutics are now widely used to promote wound repair. [56,57]The traditional view of these ROS has been seen as the culprit responsible for causing oxidative stress and damage to cells. [58]Nevertheless, more recent data show that too much ROS in cells may be harmful, but completely eliminating ROS will prevent wounds from healing. [53,56]nder a hostile wound microenvironment (such as a diabetic wound), there is insufficient ROS detoxification or too much ROS, cells will lose their redox balance and oxidative stress occurs. [59]Excessive ROS accumulated on the wound tissue will not only lead to a significant destruction of nucleic acids, growth factors and endogenous stem cells to hinder the repair of the wound, but will trigger a strong inflammatory response, making the wound fragile. [56,57]In addition, excessive ROS seems to cause endothelial dysfunction, resulting in restricted angiogenesis. [57]Conversely, appropriate (low) concentrations of ROS can play an integral part in wound repairing, which is mainly reflected in the following aspects: 1) ROS are important in the protection of the initial wound by mediated vasoconstriction and local cell signal transduction that can induce thrombus formation; 2) local ROS signaling attracts neutrophils from local blood vessels toward the site of injury site against pathogenic microorganisms; 3) killing of pathogenic microorganisms by ROS-driven phagocytosis, providing further signals for subsequent wound responses; 4) recruitment of monocytes to the wound area to help eliminate invading pathogens; 5) stimulating endothelial cells for vascular remodeling, promoting fibroblasts to divide and migrate to form a new extracellular matrix, and accelerating the proliferation and migration of keratinocytes. [60]In the wound, ROS is like a double-edged sword, and therefore strictly regulating the content of ROS is crucial for the normal repair process. [59]However, ROS has a short half-life and is difficult to measure accurately in wounds.Therefore, it will be meaningful to explore the optimal ROS content to promote wound healing.

Diabetes
With the improvement of living standards, changes in diet and lifestyle, the incidence of diabetes has significantly increased.The treatment of diabetic wounds remains a considerable challenge. [61]Diabetes can lead to vascular neuropathy, blocked blood circulation, decreased immunity, and disrupted fluid balance, thereby reducing the body's ability to repair wounds.It is noteworthy that infection is the most important cause of deterioration of a diabetic wound. [62]There are several reasons why the diabetic wound is more susceptible to infection compared with other types of wounds.Hyperglycaemic blood provides material that guarantees the proliferation of bacteria.Hypoxia, together with other metabolic effects of hyperglycemia, adversely affect the function of neutrophils and macrophages, thereby significantly reducing autoimmunity.After the bacteria multiply rapidly in a diabetic wound, the wound expands rapidly, ulcerates, and eventually the body loses control.Once tissue necrosis occurs, sepsis may develop and septic shock, in many cases, the patients have to undergo amputation. [63]Therefore, the treatment of diabetic wounds must avoid external pollution and absorb exudate, while maintaining an environment that promotes healing.

Macrophage Polarization
It is well recognized that macrophages play a key role in the host immunological responses throughout immune-regulation, wound healing, pathological conditions, and physiologically healthy.Macrophages are conventionally divided into two main subgroups: M1 and M2, which are critical to tissue regeneration and normal wound healing (Figure 5). [22]M1 macrophages appear in the early stages of wound healing, expressing proinflammatory cytokines, including tumor necrosis factor (TNF-), interleukin (IL-6) and interleukin-1 (IL-1), that fight bacterial invasion. [64]Moreover, M1 macrophages actively participate in rapid phagocytosis and kill most pathogens.They eliminate dying neutrophils in the wound at the end of inflammation, preventing damage to normal tissues and long-term inflammation. [27,65]After the inflammatory period is over, the M1 macrophage phenotype transitions into M2 macrophages, which suppress host defense, antitumor immunity and inflammatory responses. [66]M2 macrophages secrete growth factors, including VEGF, PDGF, and fibroblast growth factors, that induce cell proliferation, migration, and matrix formation. [67]During wound remodeling, M2 macrophages release proteases, engulfing cell debris, and excessive extracellular matrix. [68]As a result, managing macrophage polarization has emerged as a promising strategy in regenerative medicine.

Others
Other factors that affect wound healing include age, nutritional status, smoking, and alcohol. [44]Age: the deterioration of wound tissue repair ability in older patients is mainly manifested by decreased wound strength, slower angiogenesis and collagen deposition, delayed re-epithelialization, decreased secretion of growth factors, weaker macrophage function, increased secretion of inflammatory mediators, and enhanced platelet aggregation. [69]utritional status: malnutrition or specific nutritional deficiencies (such as insufficient protein, vitamins and trace elements) slow down wound healing.Smoking: cigarettes contain > 4000 substances, among which nicotine, carbon monoxide and hydrogen cyanide are considered to be the main factors leading to impaired wound healing. [70]On the molecular and cellular level, tobacco smoke causes tissue hypoxia, constricts blood vessels and has a negative influence on the immune response,  [22] Copyright 2020, The Royal Society.
epithelialization and fibroblastic activity.Alcohol: alcohol will dilate local capillaries, which is not conducive to wound hemostasis.Alcohol also reduces resistance and increases the risk of infection.Wound healing is simultaneously affected by multiple non-exclusive factors, so the actual situation should be fully considered when designing dressings and treatments.

Real-Time Monitoring of pH
Real-time monitoring of pH is of great significance in wound therapy because pH is an important indicator of wound status and is closely related to protease activity, angiogenesis, and bacterial infection. [71]Normally, healthy skin or healing wounds has a slightly acidic environment (pH: 4−6) due to the amino acids and fatty acids excreted by keratinocytes, which can inhibit the reproduction of bacteria. [72]However, most of the infected and chronic wounds are more alkaline, with a pH that fluctuates between 7 and 9, sometimes rising to 10 (Figure 6A), [73] indicating the possibility of infections due to bacterial incursion and colonization.Hence, the large differentials in wound pH could be a meaningful indicator to provide early alert of infection risks, making realtime pH monitoring an effective means to clarify wound status.Currently, real-time monitoring of pH levels in wounds can be realized in three different methods, including: electrochemical method, [74] colorimetric method, [75] and fluorescent method. [76]sing electrochemical methods to detect wound pH has the advantages of being sensitive, real-time, and stable, and is accepted by most people. [77]Recently, Tang et al. [78] reported an integrated electrochemical pH, temperature, and glucose sensor array within the self-healing elastomer for real-time monitoring of pH, temperature, and glucose in wound area, providing timely and reliable information of wound status (Figure 6B).In this sensing system, the pH sensors consist of a dual-electrode system containing a common Ag/AgCl reference electrode and a polyaniline working electrode.The in vivo experiments showed a significant difference in pH between uninfected and infected wounds, mainly due to the attraction of leukocytes to the injury site during the inflammatory phase.Colorimetry is a simple and direct method for continuous monitoring of wound pH, which does not require complicated instruments and steps, and has become the most popular pH monitoring method. [79]Mirani et al. [75] developed an advanced multifunctional dressing capable of colorimetric measurement pH changes at the wound site (Figure 6C).The built-in pHresponsive dye was able to measure spatial changes in pH within wounds that could be caused by different bacterial infections with accuracy comparable to commercially available systems.To improve the accuracy of colorimetry, Zhang et al. [76] presented a fluorescent/colorimetric integrated quantum dots-phenol red hydrogels, aiming to simultaneously detect the pH variation of the wound status in a real-time manner (Figure 6D).The hybridization of CQDs (fluorescent method) and phenol red (colorimetric method) with the hydrogels enables ultra-sensitive fluorescent and colorimetric responsiveness of wound pH signals to reflect the dynamic wound status.Additionally, these visual images can be captured by a smartphone and converted into pH data, allowing real-time assessment of wound dynamic conditions remotely.Real-time monitoring of pH.A) Differences in pH between healthy skin and chronic infected or chronic wounds.Reproduced with permission. [73]Copyright 2021, Elsevier.B) Integrated electrochemical pH, temperature, and glucose sensor array within the multifunctional wound dressing for real-time monitoring of pH in wound area.Reproduced with permission. [78]Copyright 2021, Wiley-Blackwell.C) Working principle and optical image of color-changing pH sensors.Reproduced with permission. [75]Copyright 2017, Wiley VCH.D) Smartphone-guided visible color and fluorescent signals for real-time pH monitoring.Reproduced with permission. [76]Copyright 2021, Wiley-VCH.
Despite the current significant progress in real-time pH detection of wound, the practical application of wound pH sensors still needs to solve many bottlenecks.First, due to subtle changes in wound pH when early infection occurs, it poses a great challenge to the sensitivity of the sensor and the accuracy of the infection identification threshold.Second, all pH sensors require detection in liquid environments, and limited availability of wound fluid in healing or dry wounds limit their usefulness.Finally, a large percentage of pH sensors suffer from chemical leaching, biocompatibility, external contamination, and calibration issues.

Real-Time Monitoring of Biofilms
The formation biofilm is a challenging problem in wound care, hindering the healing of 6% of acute wounds and 60% of chronic wounds. [9]Many bacteria that cause infection secrete polysaccharide matrices that form a biofilm adhering tightly to the wound.Biofilm formation on wounds is a dynamic process, and there is current evidence that bacteria can generate stable biofilms on the wound bed within 24 h. [80]These biofilms reduce the efficiency of killing bacteria by external factors, e.g., immune cells and antibiotics.They are a key factor in the leading of wounds to be chronic, and create one of the most difficult problems in the treatment of wound infections. [51]Therefore, it is of great significance to accurately detect the biofilm formed at the wound, especially in the early stage of its formation.Although biofilms produce clinical symptoms such as clear tissue fluid, necrotic tissue, pale wound surfaces, and yellow exudate, bacterial aggregates in wound biofilms are typically less than 100 mm in size and cannot be identified by the unaided eye. [81]There are three main traditional biofilm detection methods, namely imaging detection, Figure 7. Real-time monitoring of biofilms.A) Test procedure for biofilm sensors, with fluorescence was observed in sensor tubes containing different strains under UV irradiation.Reproduced with permission. [45]Copyright 2020, American Chemical Society.B) Schematic illustration and action mechanism of polymer sensor for biofilm detection.Reproduced with permission. [85]Copyright 2019, American Chemical Society.C) Detection mechanism of porphyrin-producing wound bacteria and real-time fluorescence images of different strains.Reproduced with permission. [86]Copyright 2020, Future Medicine Ltd. microbiological detection, and molecular detection. [82]However, these techniques generally require special equipment and trained staff, and suffer from low accuracy, long time, and high cost.Fortunately, biofilms in wounds produce quantifiable biomarkers that can be used to identify the presence of biofilms at sites of infection with high precision. [83]Sensors developed based on these biomarkers can be used to identify single or multiple pathogenic bacteria present in biofilms, thereby facilitating real-time assessment of wound status and selection of targeted treatments to improve clinical outcomes.
In pathogenic wound biofilms, virulence factors expressed by bacteria (such as rhamnolipids from Pseudomonas aeruginosa) can modulate the host immune response and lead to tissue rupture. [84]Thet et al. [45] synthesized a sensor comprising lipo-somes encapsulated with self-quenching fluorescent dyes for the detection of virulence factors expressed by bacteria in the early stages of biofilm formation (Figure 7A).The main working mechanism of the sensor is that the virulence factors released by the bacteria break down the liposomes and release the dyes in them, causing the color of the sensor to change from yellow to green, indicating the occurrence of infection.The specific operation is to place the swab in the liposome suspension after wiping the biofilm and incubate it at body temperature for one hour, and the population density of the pathogen in the biofilm model was quantified with the colorimetric reaction.Ngernpimai et al. [85] described a bacterial biofilm sensing platform based on poly(oxanorborneneimide) polymers, which enables selective multivalent interactions with biofilm substrates resulting in distinct changes in fluorescence patterns, providing speciesbased biofilm characteristics (Figure 7B).This sensor platform successfully identified biofilms formed by single pathogenic and nonpathogenic bacteria, as well as biofilms formed by mixed bacteria.Jones et al. [86] demonstrated the visualization of porphyrinproducing wound bacteria using real-time fluorescence imaging (Figure 7C).Focusing on specific wound pathogens and bacteria detected from biofilms, this study investigated the fluorescent properties of 4 yeast species and 32 bacteria under 405 nm violet light illumination.The experimental results showed that a total of 1/4 yeast species, 28/32 bacteria, and monomicrobial biofilms can produce porphyrins and perform fluorescence imaging under the excitation of violet light.Taken together, these indicative biomarkers provide great convenience for the precise detection of wound biofilms, especially in chronic non-healing infected wounds.

Real-Time Monitoring of Electrophysiological Signals
The physiological state of the wounded has a non-negligible impact on wound healing, and it has also become an entry point for doctors to analyze and diagnose pathological information. [10,87]or example, proper exercise, mental stability, and normal heart rate can help the wound recover quickly.Various physiological signals (electrocardiography, electromyography, electroencephalography, etc.) are generated in the physiological process of the human body (Figure 8A). [88]These physiological signals can be captured by electrodes to reflect the real-time physiological state of organs and tissues, and play an irreplaceable role in monitoring health conditions and evaluating clinical treatment.Depending on the application scenario, these electrodes can be divided into epidermal electrodes and invasive electrodes.Epidermal electrodes can noninvasively collect signals from the epidermis in various forms, such as fabrics, [89] tattoos, [90] and patches. [91]They do not require complex surgical implantation and have low barriers to use.Disadvantageously, epidermal electrodes must overcome the adverse effects of human skin (subcutaneous tissue, dermis, epidermis), especially the stratum corneum with high impedance.Invasive electrodes can record relatively accurate electrograms with high temporal and spatial resolution, and the implanted parts include limb muscles, heart, spinal cord, and brain. [92]Excellent invasive electrodes must ensure stable signal acquisition under the influence of physiological activities, and avoid secondary damage to the human body due to immune reactions or toxicity as much as possible.
Yan et al. [87] reported an effective strategy to synthesize cellulose bio-nanosheets assembled biostable hydrogel with good mechanical flexibility, cell/tissue affinity, and conductivity (Figure 8B,C).Due to its combination of biostable mechanical, sensitivity, and conductivity properties, the nanosheet-assembled hydrogel is well suited for epidermal bioelectronics to stably monitor electromyogram, and electrocardiogram.After the nanosheetassembled hydrogel was adhered to the volunteer's arm, the electromyogram that matched the command to contract or relax the arm could be accurately collected by a multichannel physiological signal acquisition and process system.Most importantly, even immersion in PBS for 30 days, the nanosheet-assembled hydrogel was able to detect electromyogram reproducibly and sta-bly.After integrating the nanosheet-assembled hydrogel with a wireless cardiac monitoring system, the volunteers' electrocardiogram before and after swimming were able to be remotely recorded and displayed on their mobile phones.Biological and mechanical mismatches, and unstable adhesion between invasive electrodes and implant sites, generally trigger a host immune response, leading to inaccurate signals, and potential misdiagnosis.Wang et al. [93] developed a bioadhesive ultrasoft brainmachine interface by integrating a hydrogel with flexible microcircuits for immune-evasive and conformal contact with brain tissue (Figure 8D).The catechol groups in the hydrogel can form a variety of covalent and non-covalent bonds, which endow the hydrogel with strong adhesion to brain tissue and can be tightly integrated with metal circuits.After in vivo implantation, the hydrogels exhibited immune evasion capabilities by suppressing inflammation and foreign body response mitigation strategies, actively preventing fibrous tissue encapsulation, and ensuring reliable signal transduction.This conductive, ultrasoft, bioadhesive, and immune-evasive, hydrogel-integrated brain-machine interfaces strategy provides new opportunities for long-term and accurate in vivo signal acquisition of invasive electrodes.

Real-Time Monitoring of Temperature
Temperature has been recognized as one of the twelve classic indicators for assessing wound status in clinical practice. [94]Temperature variation not only affect a series of chemical and enzymatic reactions during wound repair, but also serve as one of the indicators of inflammation and infection. [95]Typically, a decrease in local temperature indicates wound vasoconstriction, likely leading to wound ischemia and threatening wound healing.Conversely, localized vasodilation increases wound temperature and provides much-needed oxygen and nutrients to the wound site to facilitate repair.The temperature of a normal wound is usually higher than 37.8 °C, but not significantly higher than the temperature around the wound.Large increases in wound temperature (>2.2 °C) are an early sign of inflammation and infection, and are eight times more likely to have moderate or severe bacterial infection. [96]Therefore, real-time monitoring of wound temperature can be used as an effective means to sense wound status.Based on different sensing principles, several sensors are currently available for real-time monitoring of wound temperature, such as resistance temperature sensors, infrared thermal imaging, and thermochromic sensors.
Currently, portable infrared camera-based devices that provide objective thermal images of the wound site play an increasingly important role in wound monitoring. [97]Infrared camera systems with smartphones are also widely used in the field of wound thermal imaging technology to generate high-resolution 2D thermal images to further improve the efficiency of wound monitoring and treatment. [98]Infrared thermal imaging has the advantage of being easy to use and non-contact, allowing doctors, patients, and caregivers to measure wound size and temperature at any time.However, since the infrared sensor-based thermal imaging technology will be interfered by the wound dressing, the wound dressing must be removed during use, which may cause secondary damage to the wound.To remedy the above-mentioned drawbacks of infrared thermography, a thermochromic sensor with  [88] Copyright 2021, Wiley VCH.B) nanosheet-assembled hydrogel for integrated circuits and electromyogram signal detection.C) Electrocardiogram recorded by the nanosheet-assembled hydrogel integrated wireless cardiac monitoring system before and after swimming in volunteers.Reproduced with permission. [87]Copyright 2021, Wiley-VCH .D) Schematic illustration and photograph of brain-machine interface with immune-evasive, bioadhesive, ultrasoft, conductive, and transparent properties.Reproduced with permission. [93]Copyright 2022, Elsevier.
temperature visualization capability provides a promising solution, which can reflect the temperature of the wound site in real time after seamless contact with the skin. [99]By embedding thermochromic microcapsules in polyvinyl alcohol/polyurethane, He et al. [100] fabricated a thermochromic film that could change color with temperature changes (Figure 9A).On this basis, a simple and portable temperature colorimeter is designed, which can quickly and accurately understand the body surface tempera-ture by comparing the color of the standard color card and the thermochromic film (Figure 9B).Therefore, this membrane has great potential as a wearable temperature sensor for real-time wound temperature response.Among all methods, the resistance temperature sensor has stronger accuracy and practicality than other measurement methods. [101]Wang et al. [102] reported a selfhealable multifunctional electronic tattoos that shows high sensitivity to temperature variation (Figure 9C).We note that this  [100] Copyright 2019, Elsevier BV.C) Fabrication process of self-healable multifunctional electronic tattoos and relative resistance changes in different scenarios.Reproduced with permission. [102]Copyright 2019, Wiley-VCH.
resistance temperature sensor has the advantages of high sensitivity, fast response, and long-term stability, which will bring more opportunities for real-time monitoring of wound temperature.

Real-Time Monitoring of ROS
Although ROS play crucial roles in the normal healing of wounds, their overexpression in chronic wounds severely affects vital tissue-repair processes and delays the entry into the proliferative phase. [103]Therefore, real-time monitoring of the ROS concentration at the wound site will be meaningful to explore the optimal ROS content to promote wound healing.As far as we know, the current real-time monitoring of ROS in vivo mainly adopts commercial ROS detection kit (DCFH-DA) [104] or selfmade ROS-sensitive materials. [105]After entering cells, DCFH-DA is hydrolyzed by esterase to generate DCFH, which is oxidized in the presence of ROS to generate fluorescent DCF.After in situ injection of DCFH-DA into wounds, detection of fluorescent signals from DCF using an in vivo imaging system provides real-time insight into ROS content in wound cells (Fig- ure 10A). [106]However, the potential toxicity and complex imaging system seriously hinder the application of this monitoring method in humans.To this end, Chen et al. [105] proposed a sensing strategy where the recovery of oxidation-triggered fluorescence from dye-modified PSi was used as an indicator of ROS in wound.In this method, the fluorescence resonance energy transfer action caused the "dark" porous Si particles to quench the loaded luminous dye.The surface of porous Si particles was oxidized forming an insulating oxide layer in the presence of ROS, which prevented the transmission of energy between Si and dye molecules.Therefore, the fluorescence of dye molecules returned in reaction to ROS that were present in the wound (Figure 10B,C).At present, there are few researches on real-time monitoring of ROS in wounds, which has huge research value and development space.

Wearable Sensors for Wound Monitoring
Compared with the bulkiness and inefficiency of traditional wound monitoring devices, wearable sensors offer the advantages of light weight, low cost, flexibility, and simplified operation. [107]The growing field of wearable sensors enables a great potential for wound monitoring, notably in terms of improving patient compliance and introducing novel approaches to wound therapy. [108]The new generation of wearable sensors have been developed for accurate, rapid and non-invasive measurement of wound biomarkers and provide 24/7 wound monitoring.For instance, Lou et al. [11] engineered an wearable temperaturesensing system composed of data processing circuit, power manager circuit, temperature sensor STH21, and received data from the sensor through a smartphone, which can display and analyze the wound temperature in real time.Furthermore, integrated closed-loop wearable wound sensors have attracted great research interest due to their advantages in improving treatment efficiency and reducing patient intervention. [8,109]To achieve this goal, Xu  [106] Copyright 2018, American Chemical Society.Experimental mechanism B) and structural schematic diagram C) of the oxidation-induced luminescence color change of CIP-LuPSi.Reproduced with permission. [105]Copyright 2017, American Chemical Society.et al. [8] developed a battery-free, fully integrated, wearable wireless smart wound dressing for closed-loop wound management.This system enables real-time monitoring of wound uric acid, pH, and temperature to indicate bacterial infection, and delivers antibiotics to provide on-demand infection treatment.

Clinical Translation of Real-Time Wound Sensors
The clinical translation of a real-time wound sensors necessitates a comprehensive procedure in which its efficacy and safety are carefully analyzed.The most major obstacle to moving novel materials from the laboratory to the clinic is the accompanying high costs and lengthy schedule, even though the meticulous and tight regulatory process is absolutely necessary to protect patient safety. [110]Moreover, in order to cover the expenditures of product development and regulatory approval, innovative sensor designs need be financially lucrative.Particularly as a commercial product, bad features of the sensor design that might significantly raise its development expenses (such as manufacturing, sterilization, or storage) or lessen its appeal to investors and can significantly lower the chance of regulatory clearance. [111]Although such requirements are frequently not appreciated for academic research, it is crucial for materials scientists to be conversant with the regulatory routes and the relevant needs at the early-stage of product development to reduce translation hurdles. [112]Current clinical assessment of wounds relies on visual inspection to qualitatively evaluate characteristics such as bacterial infection, reepithelialization, and granulation tissue formation.Quantitative analysis of biochemical parameters in wounds often relies on downstream laboratory assays such as wound microbial culture and enzyme-linked immunosorbent assay. [113]In the fore-seeable future, highly integrated wound monitoring systems with versatility, wearability, and real-time performance will be gradually applied to the clinic, thereby completely subverting traditional wound management methods.

Opinion and Discussion
Currently, there are still several bottlenecks that need to be resolved in the field of real-time wound monitoring.The first challenge is to achieve real-time monitoring of abundant biomarkers in a limited wound area with the designed sensors.The currently developed sensors enable real-time monitoring of a limited number of targets, such as several common analytes (eg, uric acid, glucose) and physical signals (eg, temperature and pressure).Future wound biosensors should be integrated and miniaturized to expand the detection range of biomarkers and provide dynamic real-time data based on changes in wound conditions.In addition, establishing a one-to-one correspondence between biomarker data and wound status is also of great significance for sensor design.Another challenge for wound sensors is that they require a certain amount of wound fluid to get accurate data.The inability to control wound fluid production and the amount collected by the sensor can lead to errors in sample concentration and unreliable data.Therefore, sensors should be combined with advanced wound fluid collection devices, such as microneedle arrays, microfluidic channels, and other water-absorbing materials, to achieve accurate detection of wound biomarkers.Last but not least, the purpose of developing wound sensors should be to advance clinical wound therapy and not just stay at the laboratory or animal model stage.

Conclusions and Outlook
In this review, we highlight the healing analysis and real-time status awareness of wound, aiming to deepen the existing understanding of wounds and guide wound treatment towards realtime precision.First, acute, and chronic wounds are described in detail regarding their characteristics and causes.Afterward, the four main stages of wound healing are introduced in detail, and the changes in each stage are analyzed for both their macroscopic performance and their microscopic mechanisms (changes at the tissue, cellular and molecular levels).Furthermore, the most significant factors (infection, ROS, diabetes, macrophage polarization, and age) that affect wound healing are discussed.Finally, we elaborate on the current state-of-the-art in wound state perception, with a particular attention to the real-time pH, biofilms, electrophysiological signal, temperature, and ROS monitoring.
Over the past few decades, extensive academic and industrial research has led to rapid advances in wound therapy.Many theoretical knowledge of wound healing has been refined, and a series of wound state perception methods have been developed on this basis.However, despite these advances and successes, there are many opportunities for further improvement.On one hand, a systematic summary of the theoretical knowledge of wound healing is required as a theoretical basis for wound treatment and monitoring.On the other hand, combining wireless technologies (e.g., Bluetooth, near-field communication, and radio frequency identification) to transmit the monitored wound information is a promising approach for real-time and personalized perception of wound status.

Figure 2 .
Figure 2. Mechanisms of epithelial fusion and repair.A) Epithelial wound closure staining.B) Cell-shape changes and relative movement during repair of an epithelial tissue culture wound.C) Schematic diagram of adhesion between two confronting epithelial cells.D) Two adjacent emigrating growth cones of ganglia sensory neurons using filopodial extensions to search for the correct target cells.E) Sensory filopodia expressed during Drosophila dorsal closure.F) Transmission electron micrograph images showing the Drosophila dorsal closure where two epithelial cells meet and subsequently create mature adherens junctions.Reproduced with permission.[28]Copyright 2001, Springer Nature.

Figure 3 .
Figure 3. Macroscopic overview of vessel formation.A) The process of vasculogenic assembly, artery/vein differentiation, and lumen formation.B)Steps of vessel sprouting: sprouting, guidance, branching, anastomoses, and lumen formation.C) The process of vascular remodeling, from a primitive toward a stabilized and mature vascular plexus (from left to right).Reproduced with permission.[37]Copyright 2011, Cell Press.

Figure 4 .
Figure 4. Formation of biofilm in wounds and the effect of ROS on wound healing.A) Representative images of infected wounds.Reproduced with permission.[50]Copyright 2021, Mary Ann Liebert Inc. B) Bottom cross-section image and color stereo image of growing Vibrio cholerae biofilm cluster at 18 h.C) Schematic diagram of the steps in Vibrio cholerae biofilm formation.The matrix is pink and the cells are yellow.D) Changes of verticalization time after using chemicals to control the cell length of Vibrio cholerae.Reproduced with permission.[52]Copyright 2019, Cell Press.E) Influence of ROS to modulate key events that occur during hemostasis, inflammation, proliferation, and dermal remodeling phases.Reproduced according to the terms of the CC BY license.[53]Copyright 2018, The Authors, published by MDPI.

Figure 6 .
Figure 6.Real-time monitoring of pH.A) Differences in pH between healthy skin and chronic infected or chronic wounds.Reproduced with permission.[73]Copyright 2021, Elsevier.B) Integrated electrochemical pH, temperature, and glucose sensor array within the multifunctional wound dressing for real-time monitoring of pH in wound area.Reproduced with permission.[78]Copyright 2021, Wiley-Blackwell.C) Working principle and optical image of color-changing pH sensors.Reproduced with permission.[75]Copyright 2017, Wiley VCH.D) Smartphone-guided visible color and fluorescent signals for real-time pH monitoring.Reproduced with permission.[76]Copyright 2021, Wiley-VCH.

Figure 8 .
Figure 8. Real-time monitoring of electrophysiological signals.A) Origins of different electrophysiological signals, from left to right are: electrocardiography, electroencephalography, and electromyography.Reproduced with permission.[88]Copyright 2021, Wiley VCH.B) nanosheet-assembled hydrogel for integrated circuits and electromyogram signal detection.C) Electrocardiogram recorded by the nanosheet-assembled hydrogel integrated wireless cardiac monitoring system before and after swimming in volunteers.Reproduced with permission.[87]Copyright 2021, Wiley-VCH .D) Schematic illustration and photograph of brain-machine interface with immune-evasive, bioadhesive, ultrasoft, conductive, and transparent properties.Reproduced with permission.[93]Copyright 2022, Elsevier.

Figure 9 .
Figure 9. Real-time temperature monitoring.A) Colorimetric response of the colorimetric temperature sensor.B) applications of temperature colorimeter in different locations of body surface.Reproduced with permission.[100]Copyright 2019, Elsevier BV.C) Fabrication process of self-healable multifunctional electronic tattoos and relative resistance changes in different scenarios.Reproduced with permission.[102]Copyright 2019, Wiley-VCH.

Figure 10 .
Figure 10.Real-time monitoring of ROS.A) Schematic illustration of in vivo detection of ROS using DCFH-DA.Reproduced with permission.[106]Copyright 2018, American Chemical Society.Experimental mechanism B) and structural schematic diagram C) of the oxidation-induced luminescence color change of CIP-LuPSi.Reproduced with permission.[105]Copyright 2017, American Chemical Society.