Systems and methods for using nanomembrane electronics - Patents.com

Abstract

Described herein is a wireless nanomembrane non-invasive system that integrates skin-wearable printed sensors and electronics, and methods that can be used to monitor electrophysiological parameters of a subject or to identify therapeutic agents. The system can include a wearable device that includes a skin-wearable printed sensor and electronics for real-time continuous monitoring of the electrophysiological parameters of a subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 194,113, filed May 27, 2021, which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. R01AR071397 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Animal models can provide invaluable information in the pursuit of scientific and medical knowledge and the development of new drugs and treatments, specifically in the preclinical testing of such research and development. An animal model is a non-human species used in biomedical and drug research because it can mimic aspects of biological processes or diseases found in humans. Animal models (e.g., mice, rats, zebrafish, and others) share similarities with humans in their anatomy, physiology, or response to pathogens, which allows researchers to extrapolate the results of animal model studies to better understand human physiology and disease. By using animal models, researchers can perform experiments that are not practical or ethically prohibited in humans.

[0004] In most preclinical studies, the animal model will be co-introduced or periodically co-introduced with the drug or treatment of interest, and then euthanized after a predefined period of time to assess the physiological response of the animal model.

[0005] There are advantages to extracting other clinically relevant information from animal models in a non-invasive manner. Summary of the Invention

[0006] Described herein is a wireless nanomembrane non-invasive system integrating a skin-wearable printed sensor and electronics, and a method that can be used to monitor electrophysiological parameters of a subject or to identify therapeutic agents. The system can include a wearable device including a skin-wearable printed sensor and electronics for real-time continuous monitoring of the electrophysiological parameters of a subject.

[0007] The described methods can monitor an electrophysiological parameter of a subject. The methods can include acquiring a signal from a wearable device described herein and using the acquired signal to assess a disease progression in the subject, an injury in the subject, or any combination thereof to provide real-time continuous monitoring of the electrophysiological parameter of the subject.

[0008] The described methods can identify a therapeutic agent, the methods can include contacting a wearable device including a skin-wearable printed sensor with the skin of a subject, acquiring a signal from the wearable device on the skin of the subject, administering an agent of interest to the subject, acquiring a signal from the wearable device on the skin of the subject after administration of the agent of interest, comparing the signals of the subject before and after administration of the agent of interest, and analyzing results from the comparing step to assess a physiological parameter of the subject, wherein the physiological parameter provides an indication that the agent of interest is a therapeutic agent.

[0009] The described methods can identify diagnostic agents, and can include contacting a wearable device comprising a skin-wearable printed sensor with the skin of a subject, acquiring a signal from the wearable device on the skin of the subject, administering the agent of interest to the subject, acquiring a signal from the wearable device upon subsequent administration of the agent of interest to the skin of the subject, comparing the signals of the subject before and after administration of the agent of interest, and analyzing the results from the comparing step to evaluate a physiological parameter of the subject, the physiological parameter providing an indication that the agent of interest is a diagnostic agent. In some embodiments, the wearable device can include a system described herein.

[0010] In some embodiments, the subject can be an animal model also described herein. The animal model can include an animal subject with a wearable device described herein on the skin of the animal subject, where the animal subject has been administered an agent of interest, has been subjected to injury, or any combination thereof. In some embodiments, the animal subject has been administered an agent of interest. In some embodiments, the animal subject has been subjected to injury. In some embodiments, the animal subject has been administered an agent of interest and has been subjected to injury.

[0011] In some embodiments, the injury can include induced masseter muscle injury. In some embodiments, the animal model can be a craniofacial VML model. In some embodiments, the craniofacial VML model can include an animal subject that is subjected to biopsy punch induced masseter muscle injury. In some embodiments, the animal model exhibits impaired muscle regeneration and imbalance of muscle resident stem cell activity. [Brief description of the drawings]

[0012] [Figure 1] 1 shows an exemplary system configured for real-time, continuous monitoring of an animal model, according to an illustrative embodiment. [Diagram 2]1A and 1B collectively show an exemplary method of operating a wearable sensor system for evaluating a drug or treatment of interest (e.g., to identify or confirm a therapeutic agent or an effective dosage thereof) by monitoring a disease, injury, or condition progression in a subject, according to an illustrative embodiment. [Figure 3A] 2 shows the exemplary skin-wearable sensor system of FIG. 1, in accordance with an illustrative embodiment. [Figure 3B] 2 shows the exemplary skin-wearable sensor system of FIG. 1, in accordance with an illustrative embodiment. [Figure 3C] 2 shows the exemplary skin-wearable sensor system of FIG. 1, in accordance with an illustrative embodiment. [Figure 3D] 2 shows the exemplary skin-wearable sensor system of FIG. 1, in accordance with an illustrative embodiment. [Figure 4] 1 illustrates an example circuit and / or layout of a sensor system, according to an illustrative embodiment. [Figure 5A] 2 shows a printing process for graphene electrodes and a micro-fabrication process for thin-film-based circuits, e.g., for the sensor system of FIG. 1, according to an illustrative embodiment. [Figure 5B] 2 shows a printing process for graphene electrodes and a micro-fabrication process for thin-film-based circuits, e.g., for the sensor system of FIG. 1, according to an illustrative embodiment. [Figure 6A] We outline a study to evaluate real-time functional testing of craniofacial VML in mice using wireless nanomembrane electronics. [Figure 6B] We outline a study to evaluate real-time functional testing of craniofacial VML in mice using wireless nanomembrane electronics. [Figure 6C] We outline a study to evaluate real-time functional testing of craniofacial VML in mice using wireless nanomembrane electronics. [Figure 6D] We outline a study to evaluate real-time functional testing of craniofacial VML in mice using wireless nanomembrane electronics. [Figure 6E] We outline a study to evaluate real-time functional testing of craniofacial VML in mice using wireless nanomembrane electronics. [Figure 7A] FIG. 7 shows the results of characterization of the graphene film electrodes and wireless flexible circuitry in the tests. [Figure 7B] FIG. 7 shows the results of characterization of the graphene film electrodes and wireless flexible circuitry in the tests. [Figure 7C] FIG. 7 shows the results of characterization of the graphene film electrodes and wireless flexible circuitry in the tests. [Figure 7D] FIG. 7 shows the results of characterization of the graphene film electrodes and wireless flexible circuitry in the tests. [Figure 7E] FIG. 7 shows the results of characterization of the graphene film electrodes and wireless flexible circuitry in the tests. [Figure 7F] FIG. 7 shows the results of characterization of the graphene film electrodes and wireless flexible circuitry in the tests. [Figure 7G] FIG. 7 shows the results of characterization of the graphene film electrodes and wireless flexible circuitry in the tests. [Figure 7H] FIG. 7 shows the results of characterization of the graphene film electrodes and wireless flexible circuitry in the tests. [Figure 8A] 1 shows a study to demonstrate craniofacial VML and defective masseter muscle regeneration. [Figure 8B] 1 shows a study to demonstrate craniofacial VML and defective masseter muscle regeneration. [Figure 8C] 1 shows a study to demonstrate craniofacial VML and defective masseter muscle regeneration. [Figure 8D] 1 shows a study to demonstrate craniofacial VML and defective masseter muscle regeneration. [Figure 8E] 1 shows a study to demonstrate craniofacial VML and defective masseter muscle regeneration. [Figure 8F] 1 shows a study to demonstrate craniofacial VML and defective masseter muscle regeneration. [Figure 9A] We demonstrate the operation of a wireless, wearable EMG system for assessing functionality of the VML-injured masseter muscle. [Figure 9B]We demonstrate the operation of a wireless, wearable EMG system for assessing functionality of the VML-injured masseter muscle. [Figure 9C] We demonstrate the operation of a wireless, wearable EMG system for assessing functionality of the VML-injured masseter muscle. [Figure 9D] We demonstrate the operation of a wireless, wearable EMG system for assessing functionality of the VML-injured masseter muscle. [Figure 9E] We demonstrate the operation of a wireless, wearable EMG system for assessing functionality of the VML-injured masseter muscle. [Figure 9F] We demonstrate the operation of a wireless, wearable EMG system for assessing functionality of the VML-injured masseter muscle. [Figure 9G] We demonstrate the operation of a wireless, wearable EMG system for assessing functionality of the VML-injured masseter muscle. [Figure 10A] We demonstrate the operation for monitoring functional recovery after transplantation of the VML-injured masseter muscle. [Figure 10B] We demonstrate the operation for monitoring functional recovery after transplantation of the VML-injured masseter muscle. [Figure 10C] We demonstrate the operation for monitoring functional recovery after transplantation of the VML-injured masseter muscle. [Figure 10D] We demonstrate the operation for monitoring functional recovery after transplantation of the VML-injured masseter muscle. [Figure 10E] We demonstrate the operation for monitoring functional recovery after transplantation of the VML-injured masseter muscle. [Figure 10F] We demonstrate the operation for monitoring functional recovery after transplantation of the VML-injured masseter muscle. [Figure 10G] We demonstrate the operation for monitoring functional recovery after transplantation of the VML-injured masseter muscle. [Figure 11A] The results of computational dynamics simulations are shown. [Figure 11B] The results of computational dynamics simulations are shown. [Figure 12A] 4 shows the results of filtered EMG signals from a mouse after VML injury. [Figure 12B]4 shows the results of filtered EMG signals from a mouse after VML injury. [Figure 12C] 4 shows the results of filtered EMG signals from a mouse after VML injury. [Figure 12D] 4 shows the results of filtered EMG signals from a mouse after VML injury. [Figure 13A] 1 shows the results of stem cell dysregulation in the masseter muscle 3 days after VML injury. [Figure 13B] 1 shows the results of stem cell dysregulation in the masseter muscle 3 days after VML injury. [Figure 13C] 1 shows the results of stem cell dysregulation in the masseter muscle 3 days after VML injury. [Figure 13D] 1 shows the results of stem cell dysregulation in the masseter muscle 3 days after VML injury. [Figure 13E] 1 shows the results of stem cell dysregulation in the masseter muscle 3 days after VML injury. [Figure 14] Shown is the filtered EMG signal of an uninjured mouse during feeding. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Several references, which may include various patents, patent applications, and publications, are cited in the reference list and discussed in the disclosure provided herein. Citation and / or discussion of such references is provided merely to clarify the description of the present disclosure and is not an admission that any such reference is "prior art" to any aspect of the present disclosure described herein. For purposes of notation, "[n]" corresponds to the nth reference in the list. All references cited and discussed herein are incorporated herein by reference in their entirety and to the same extent as if each reference was incorporated by reference separately.

[0014] Several references, which may include various patents, patent applications, and publications, are cited in the reference list and discussed in the disclosure provided herein. Citation and / or discussion of such references is provided merely to clarify the description of the disclosed technology, and is not an admission that any such reference is "prior art" to any aspect of the disclosed technology described herein. For purposes of notation, "[n]" corresponds to the nth reference in the list. For example, [1] refers to the first reference in the list. All references cited and discussed herein are incorporated herein by reference in their entirety and to the same extent as if each reference was incorporated by reference separately.

[0015] definition It should also be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" or "approximately 5" one particular value, and / or to "about" or "approximately" another particular value. When such a range is expressed, other exemplary embodiments include the one particular value and / or the other particular value.

[0016] "Comprising" or "containing" or "including" means that at least the named compound, element, particle, or method step is present in a composition or article or method, but does not exclude the presence of other compounds, materials, particles, or method steps, even if other such compounds, materials, particles, or method steps have the same function as the named one.

[0017] As used herein, the terms "may," "optionally," and "optionally may" are used interchangeably and are meant to include cases where the condition occurs as well as cases where the condition does not occur. Thus, for example, a statement that a formulation "may include an excipient" is meant to include cases where the formulation includes an excipient as well as cases where the formulation does not include an excipient.

[0018] It is understood that when combinations, subsets, groups, and the like of elements (e.g., combinations of components in a composition or combinations of steps in a method) are disclosed, specific reference to each of the various separate and collective combinations, and permutations of these elements may not be explicitly disclosed, and each is specifically contemplated and described herein.

[0019] The term "about" as used herein means approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10%. In one embodiment, the term "about" means ±10% of the numerical value of the number with which the term is used. Thus, about 50% means within a range of 45% to 55%. Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, 4.24, and 5).

[0020] Similarly, numerical ranges recited herein by endpoints include the subranges subsumed within that range (e.g., 1 to 5 includes 1 to 1.5, 1.5 to 2, 2 to 2.75, 2.75 to 3, 3 to 3.90, 3.90 to 4, 4 to 4.24, 4.24 to 5, 2 to 5, 3 to 5, 1 to 4, and 2 to 4). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0021] As discussed herein, a "subject" may be any applicable animal, or other organism (living or dead), or other biological or molecular structure or chemical environment, and may relate to a particular component of the subject, e.g., a particular tissue or bodily fluid of the subject (e.g., human tissue within a particular area of ​​the body of a living subject), which may be in a particular location of the subject, referred to herein as an "area of ​​interest" or "region of interest."

[0022] It is to be understood that the animal may be of any of a variety of applicable types, including, but not limited to, a mammal, a veterinary animal, a livestock animal, or a pet-type animal, etc. By way of example, the animal may be a laboratory animal (e.g., rats, dogs, pigs, monkeys) specifically selected to have certain characteristics similar to humans, etc.

[0023] "Administration" to a subject includes any route of introducing or delivering an agent to a subject. Administration can be performed by any suitable route, including oral, topical, transcutaneous, transdermal, intra-articular, intra-arteriolar, intradermal, intraventricular, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion techniques), and the like. As used herein, "concurrent administration," "mixed administration," "simultaneous administration," or "administered simultaneously" means that the compounds are administered at the same point in time or essentially immediately after each other. In the latter case, the two compounds are administered close enough in time that the results observed are indistinguishable from the results achieved when the compounds are administered at the same point in time. "Systemic administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to a wide area of ​​the subject's body (e.g., more than 50% of the body), for example, through an entrance into the circulatory or lymphatic system. In contrast, "local administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to the area of ​​administration, or to an area immediately adjacent to the point of administration, and does not introduce the agent systemically in therapeutically significant amounts. For example, a locally administered agent is readily detectable in the local vicinity of the point of administration, but is undetectable or detectable in negligible amounts in distal portions of the subject's body. Administration includes self-administration and administration by another.

[0024] As used herein, an "agent of interest" may be a therapeutic agent, a diagnostic agent, or a prophylactic agent. An agent may be an organic molecule (e.g., a therapeutic agent, a drug), an inorganic molecule, a nucleic acid, a protein, an amino acid, a peptide, a polypeptide, a polynucleotide, a targeting agent, an isotopically labeled organic or inorganic molecule, a vaccine, an immunological agent, and the like. In some embodiments, the term "agent of interest" is used herein to refer to a chemical compound or composition that may have a beneficial biological effect. Beneficial biological effects include therapeutic effects, i.e., the treatment of a disorder or other undesirable physiological condition, and prophylactic effects, i.e., the prevention of a disorder or other undesirable physiological condition. These terms also encompass pharma- ceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the term "beneficial agent" or "active agent" is used, then, or when a particular agent is specifically identified, it should be understood that the term includes the agent itself, as well as pharma- ceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, complexes, active metabolites, isomers, fragments, analogs, etc. In some embodiments, an agent of interest can be an agent that has been determined to be a suitable therapeutic agent for ameliorating injury.

[0025] As used herein, the terms "beneficial agent" and "active agent" are used interchangeably herein to refer to a chemical composition or composition that has a beneficial biological effect. Beneficial biological effects include therapeutic effects, i.e., the treatment of a disorder or other undesirable physiological condition, and prophylactic effects, i.e., the prevention of a disorder or other undesirable physiological condition. These terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the term "beneficial agent" or "active agent" is used, then, or when a particular agent is specifically identified, it should be understood that the term includes the agent itself, as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, complexes, active metabolites, isomers, fragments, analogs, and the like.

[0026] "Reduction" can refer to any change that results in a lower amount of symptoms, disease, composition, condition, or activity. A substance is also understood to reduce the genetic output of a gene when the genetic output of the gene product containing the substance is less relative to the output of the gene product without the substance. A reduction can also be, for example, a change in the symptoms of a disorder, such that the symptoms are less than previously observed. A reduction can be any individual, median, or average reduction in a statistically significant amount of a condition, symptom, activity, or composition. Thus, a reduction can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% reduction, as long as the reduction is statistically significant.

[0027] "Inhibit", "inhibiting" and "inhibition" refer to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% reduction, or any amount in between, compared to native or control levels.

[0028] "Inactivate," "inactivating," and "inactivation" mean to reduce or eliminate an activity, response, condition, disease, or other biological parameter resulting from a chemical (covalent bond formation) between a ligand and its biological target.

[0029] "Reduce" or other forms of this term, such as "reducing" or "reduction", refers to a decrease in an event or characteristic (e.g., tumor growth). This is typically relative to some standard or expected value, in other words, it is relative, but it is understood that it is not necessarily to refer to a standard or relative value. For example, "reducing tumor growth" means reducing the rate of growth of a tumor compared to a standard or control.

[0030] As used herein, the term "treating" or "treatment" of a subject includes administering a drug to a subject for the purpose of preventing, curing, alleviating, relieving, relieving, altering, correcting, enhancing, ameliorating, stabilizing, or affecting a disease or disorder, or a symptom of a disease or disorder. The terms "treating" and "treatment" can also refer to reducing the severity and / or frequency of symptoms, eliminating symptoms and / or their underlying causes, preventing the occurrence of symptoms and / or their underlying causes, and ameliorating or correcting damage.

[0031] "Prevent" or other forms of the word, such as "preventing" or "prevention", mean to stop a particular event or characteristic, stabilize or delay the development or progression of a particular event or characteristic, or minimize the chance that a particular event or characteristic may occur. Prevention is typically more absolute than, for example, reduction, and therefore does not require a comparison to a control. As used herein, something can be reduced, but not prevented, but something that is reduced can be prevented. Similarly, something can be prevented, but not reduced, but something that is prevented can be reduced. When reduction or prevention is used, it is understood that the use of other words is also expressly disclosed unless specifically specified otherwise. For example, the term "prevent" or "suppress" can refer to a treatment that arrests or delays the onset of a disease or condition, or reduces the severity of a disease or condition. Thus, if a treatment can treat a disease in a subject who has symptoms of the disease, it can also prevent or suppress that disease in a subject who does not yet suffer from some or all of the symptoms. As used herein, the term "preventing" a disorder or undesirable physiological event in a subject specifically refers to preventing the occurrence of the symptoms and / or their underlying causes, and the subject may or may not exhibit an increased susceptibility to the disorder or event.

[0032] The term "effective amount" of a therapeutic agent refers to a non-toxic but sufficient amount of the beneficial agent to produce the desired effect. The amount of the beneficial agent that is "effective" will vary from subject to subject, depending on the age and general condition of the subject, the particular beneficial agent or agents. Thus, it is not always possible to specify an exact "effective amount". However, an appropriate "effective amount" in any subject case can be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, unless otherwise specified, an "effective amount" of a beneficial agent can refer to an amount that covers both a therapeutically effective amount and a prophylactically effective amount.

[0033] The "effective amount" of a drug required to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0034] As used herein, a "therapeutically effective amount" of a therapeutic agent refers to an amount that is effective to achieve a desired therapeutic result, and a "prophylactically effective amount" of a therapeutic agent refers to an amount that is effective to prevent an undesirable physiological condition. The therapeutically effective amount and prophylactically effective amount of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated, as well as the age, sex, and weight of the subject. The term "therapeutically effective amount" may also refer to the amount of therapeutic agent, or the rate of delivery of the therapeutic agent (e.g., amount over time), effective to promote a desired therapeutic effect. The exact desired therapeutic effect will vary according to the condition being treated, the tolerability of the subject, the agent and / or agent formulation being administered (e.g., potency of the therapeutic agent (drug), concentration of the drug in the formulation, etc.), as well as various other factors understood by those skilled in the art.

[0035] As used herein, a "pharmaceutical acceptable" ingredient refers to an ingredient that is not biologically or otherwise undesirable, i.e., an ingredient that can be incorporated into a pharmaceutical formulation of the present invention and administered to a subject as described herein, without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other ingredients of the formulation in which it is contained. When the term "pharmaceutical acceptable" is used to refer to an excipient, the term generally means that the ingredient has met the necessary standards of toxicological and manufacturing testing, or that it is included in the Inactive Ingredients Guide prepared by the U.S. Food and Drug Administration.

[0036] A "pharmaceutical acceptable carrier" (sometimes referred to as a "carrier") means a carrier or excipient that is generally safe and non-toxic and useful in the preparation of a pharmaceutical or therapeutic composition, and includes carriers that are acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutical acceptable carrier" can include, but is not limited to, phosphate buffered saline, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations, and materials further described herein.

[0037] As used herein, "pharmaceutical acceptable salts" are derivatives of the disclosed compounds, in which the parent compound is modified by making its inorganic and organic, non-toxic, acid or base addition salts. The salts of the compounds can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of a suitable base (such as Na, Ca, Mg, or K hydroxides, carbonates, bicarbonates, etc.) or by reacting the free base forms of these compounds with a stoichiometric amount of a suitable acid. Such reactions are typically carried out in water or an organic solvent, or a mixture of the two. In general, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are typical, where feasible. The salts of the compounds further include solvates of the compounds and solvates of the compound salts.

[0038] Examples of pharma- ceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, etc. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids, and quaternary ammonium salts. For example, conventional non-toxic acid salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like, as well as salts derived from acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, mesylic acid, esylic acid, besylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, HOOC-(CH2) n -COOH, where n is 0 to 4, or using a different acid that produces the same counterion. Additional listings of suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 17th Edition, Mack Publishing Company, Easton, Pa., p1418 (1985).

[0039] Additionally, as used herein, "pharmacologically active" (or simply "active") can refer to derivatives or analogs (e.g., salts, esters, amides, complexes, metabolites, isomers, fragments, etc.) that have the same type and approximately the same degree of pharmacological activity as the parent compound, as in "pharmacologically active" derivatives or analogs.

[0040] A "control" is a substitute control or sample used in an experiment for comparison purposes. Controls can be "positive" or "negative."

[0041] In describing the exemplary embodiments, technical terms will be used for clarity. Each term is intended to assume the broadest meaning of that term as understood by those skilled in the art and to include all technical equivalents that operate in a similar manner to achieve a similar purpose. It should also be understood that the reference to one or more steps of a method does not preclude the presence of additional or intervening method steps between those steps explicitly identified. The steps of the method may be performed in a different order than described herein without departing from the scope of the present disclosure. Similarly, it should also be understood that the reference to one or more components in a device or system does not preclude the presence of additional or intervening components between those components explicitly identified.

[0042] Exemplary System FIG. 1 shows an exemplary system 100 configured for real-time continuous monitoring of an animal model, according to an illustrative embodiment. The system 100 can be used to monitor physiological parameters or metrics of the animal model to allow for continuous evaluation of a drug or surgical treatment of interest. The system 100 can augment or replace current experimental evaluation paradigms and procedures for evaluation of therapeutic drugs or surgically induced procedures via the animal model. The system 100 is miniaturized to integrate with the animal model for portable real-time monitoring over an extended period of time.

[0043] In the example shown in FIG. 1A, the system 100 includes an animal model 102 coupled to or layered with a wireless, non-invasive, skin-wearable sensor system 104. The sensor system 104 includes a set of one or more sensors 106 (shown as 106a) for acquiring one or more biophysical signals 108 from the animal model 102. The sensors 106 can acquire time series data or channels thereof, such as inertia, acceleration, orientation, temperature, or sound. The acquired time series data of the sensors 106 can also include electromyogram (EMG), electrocardiogram (ECG), electroencephalogram (EEG), phonocardiogram, electrical potential, impedance, and acoustics. The sensors 106 can be configured as a sensor array for acquiring time series array data, e.g., time series data or images.

[0044] In the example shown in FIG. 1, the skin-wearable sensor system 104 includes a primary sensor set including a stretchable sensor 110 configured, for example, as a stretchable membrane electrode 110a, an auxiliary sensor set 112 including, for example, an inertial measurement sensor 112, as well as acquisition electronics 114, a controller 116, an energy storage module 118, and a network interface 120. The stretchable membrane sensor 110, or sensor assembly, can be attached to the underside of the skin-wearable sensor system 104, for example, via a software membrane underlayer. In some embodiments, the stretchable membrane sensor 110 can be configured as an external sensor (shown as 122 in FIG. 1) that can be attached to another area of ​​the animal model 102 and connected to the sensor system 104 via an interconnect 124. The stretchable membrane sensor 110, or sensor assembly, can be configured to have stretchable electrodes for directly acquiring the biophysical signal 108 from the animal model 102 or to have stretchable contact pads that can be coupled to an integrated sensor circuit.

[0045] The exemplary system 100 can include an ultra-thin, low-profile, lightweight, and stretchable membrane sensor based on biocompatible thin-film soft circuits (e.g., graphene) for data processing, and can provide seamless attachment to the skin of living animal models (e.g., mice or rats) without interfering with their natural behavior. In addition, the compact device integration on a soft elastomer platform can provide comfortable wearability without movement artifacts caused by cumbersome wires and rigid systems. The use of a non-invasive and ergonomic monitoring system / device allows for movement during measurements, thus enabling the system to monitor physiological responses in a natural ambulatory environment.

[0046] The skin-wearable sensor system 104 is configured to communicate via a short-range communication channel 126 with a data acquisition system 128 comprised of a network interface 130, data storage 132, and a monitoring and control module 134 (also referred to as a controller 134). The acquired data can then be analyzed in an analysis system or operation 136. The data acquisition system 128 can be a customized data storage system, a standard computing device configured with internal data acquisition hardware, or a standard computing device configured with external data acquisition hardware.

[0047] As mentioned above, the skin-wearable sensor system 104 includes auxiliary sensors, such as the inertial measurement sensor 112, that can provide inertial, acceleration, and orientation information related to a movement or activity. The inertial signals (e.g., inertial, acceleration, orientation information) of the inertial measurement sensor 112 can be used to remove noise or motion artifacts from the stretchable sensor 110.

[0048] The acquisition electronics 128 may include an analog-to-digital converter or a capacitance-to-digital converter, a transimpedance amplifier or other amplifier circuitry, appropriate filters (e.g., low-pass and / or high-pass filters), and corresponding circuitry for voltage regulation and clocks.

[0049] The controller 134 includes a processing unit, which may be a standard programmable processor that performs arithmetic and logic operations necessary for the operation of the computing device. As used herein, processing unit and processor refer to a physical hardware device that executes coded instructions to perform functions on inputs and create outputs, including, but not limited to, microprocessors (MCUs), microcontrollers, graphic processing units (GPUs), and application specific integrated circuits (ASICs). Thus, although instructions may be discussed as being executed by a processor, the instructions may be executed simultaneously, sequentially, or otherwise by one or more processors. The computing device may also include a bus or other communication mechanism for communicating information between various components of the computing device. Multiple processors may be used by the controller 134.

[0050] The data acquisition system 128 can be a customized data storage system, a standard computing device configured with internal data acquisition hardware, or a standard computing device configured with external data acquisition hardware. The data acquisition system 128 can include an interface and display for presenting one or more biophysical signals 108 from the animal model. The data acquisition system 128 can acquire and present data and record data from the multiple skin-wearable sensor systems 104 as well as from other instrumentation. In some embodiments, the monitoring and control module 134 is configured with a web services module that can curate or present one or more biophysical signals 108 via a web portal hosted by the web services module. In some embodiments, the monitoring and control module 134 is configured to interface with a cloud infrastructure to provide acquired biophysical and instrumentation data to a cloud-based analysis system or off-server via a cloud-based storage infrastructure.

[0051] Examples of biophysical signals 108 are shown in FIG. 1 as an electrocardiogram 108 a acquired via a stretchable membrane sensor 110, and accelerometer and angular acceleration measurements 108 b, 108 c acquired via an inertial measurement sensor 112.

[0052] The network interface 130 is configured to communicate between the skin-wearable sensor system 104 and the data acquisition system 128. The network interface (e.g., 120, 130) may include a universal serial bus (USB) interface, a serial interface, a wireless local area network (WLAN), or a low power chipset for a wireless transceiver such as Bluetooth, wireless USB, or other short-range communication protocol.

[0053] The energy storage module 118 is configured to provide energy to the skin-wearable sensor system 104 and the sensors. The energy storage module 118 can include rechargeable circuitry and a rechargeable battery (e.g., lithium or nickel-cadmium) that can be wirelessly charged via inductive charging operations. In some embodiments, the energy storage module 118 includes a power converter and connects to a power source via a wire connection.

[0054] Data storage 132 can be a local data store of the computing device. In some embodiments, data storage 132 is a cloud-based data store. The data store is a repository for consistently storing and managing a collection of data. In some embodiments, the data store can maintain files in a hierarchical database.

[0055] The analysis system or operations 136 may include a statistical analysis of the acquired biophysical signal 108. In some embodiments, the analysis system or operations 136 are configured to clean and filter the signal 108, for example to remove motion artifacts. In some embodiments, the statistical analysis may include machine learning based analysis.

[0056] In some embodiments, the analytical system is configured to generate models from the ML functions and use the ML functions in supervised or unsupervised machine learning operations to generate estimates (e.g., scores) for the likelihood of an effect of a drug or therapeutic of interest. In addition to the machine learning functions described above, the analytical system can be implemented using one or more artificial intelligence and machine learning operations. The term "artificial intelligence" may include any technology that allows one or more computing devices or computing systems (i.e., machines) to mimic human intelligence. Artificial intelligence (AI) includes, but is not limited to, knowledge-based, machine learning, representation learning, and deep learning. The term "machine learning" is defined herein to be a subset of AI that allows machines to acquire knowledge by extracting patterns from raw data. Machine learning techniques include, but are not limited to, logistic regression, support vector machines (SVMs), decision trees, naive Bayes classifiers, and artificial neural networks. The term "representation learning" is defined herein to be a subset of machine learning that allows machines to automatically discover the representations required for feature detection, prediction, or classification from raw data. Representation learning techniques include, but are not limited to, autoencoders and embeddings. The term "deep learning" is defined herein as a subset of machine learning that enables machines to use layers of processing to automatically discover the representations needed for feature detection, prediction, classification, etc. Deep learning techniques include, but are not limited to, artificial neural networks or multi-layer perceptrons (MLPs).

[0057] Machine learning models include supervised, semi-supervised, and unsupervised learning models. In a supervised learning model, the model learns a function that maps inputs (also known as feature(s)) to outputs (also known as targets) during training with a labeled data set (or data sets). In an unsupervised learning model, the model discovers patterns (e.g., structure, distribution, etc.) in unlabeled or labeled data sets. In a semi-supervised model, the model learns a function that maps inputs (also known as feature(s)) to outputs (also known as targets) during training with both labeled and unlabeled data.

[0058] Neural Network. An artificial neural network (ANN) is a computing system that includes multiple interconnected neurons (e.g., also referred to as "nodes"). The present disclosure contemplates that the nodes may be implemented using a computing device (e.g., a processing unit and memory as described herein). The nodes may be arranged in multiple layers, such as an input layer, an output layer, and one or more hidden layers, optionally with different activation functions. An ANN with hidden layers may be referred to as a deep neural network or a multi-layer perceptron (MLP). Each node is connected to one or more other nodes in the ANN. For example, each layer is composed of multiple nodes, and each node is connected to all nodes in the previous layer. The nodes in a given layer are not interconnected with each other, i.e., the nodes in a given layer function independently of each other. As used herein, the nodes in the input layer receive data from outside the ANN, the nodes in the hidden layer modify the data between the input layer and the output layer, and the nodes in the output layer provide the results. Each node is configured to receive an input, implement an activation function (e.g., a binary step, linear, sigmoidal, hyperbolic tangent, or rectified linear unit (ReLU) function), and provide an output according to the activation function. In addition, each node is associated with a respective weight. The ANN is trained with a data set to maximize or minimize an objective function. In some implementations, the objective function is a cost function that is a measure of the performance of the ANN during training (e.g., an error such as L1 or L2 loss), and the training algorithm adjusts the weights and / or biases of the nodes to minimize the cost function. The present disclosure contemplates that any algorithm that finds a maximum or minimum of an objective function may be used to train the ANN. Training algorithms for ANNs include, but are not limited to, backpropagation. It should be understood that the artificial neural network is provided only as an exemplary machine learning model. The present disclosure contemplates that the machine learning model can be any supervised, semi-supervised, or unsupervised learning model. Optionally, the machine learning model is a deep learning model.Machine learning models are known in the art and therefore will not be described in further detail herein.

[0059] A convolutional neural network (CNN) is a type of deep neural network that is applied, for example, to image analysis applications. Unlike traditional neural networks, each layer of a CNN has multiple nodes arranged in three dimensions (width, height, depth). A CNN can include different types of layers, for example, convolutional layers, pooling layers, and fully connected (also referred to herein as "dense") layers. Convolutional layers include a set of filters and perform the majority of the computations. Pooling layers are optionally inserted between convolutional layers to reduce computational power and / or control overfitting (e.g., by downsampling). Fully connected layers include neurons, each neuron connected to all neurons in the previous layer. Layers are stacked similarly to traditional neural networks. GCNN is a CNN adapted to operate on structured datasets such as graphs.

[0060] Other supervised learning models. A logistic regression (LR) classifier is a supervised classification model that uses a logistic function to predict the probability of a target, which can be used for classification. During training, the LR classifier is trained on a data set (also referred to herein as a "data set") to maximize or minimize an objective function, e.g., a measure of the performance of the LR classifier (e.g., an error such as an L1 or L2 loss). The present disclosure contemplates that any algorithm that finds a minimum of a cost function can be used. LR classifiers are known in the art and therefore will not be described in further detail herein.

[0061] Exemplary Methods 2A and 2B collectively show an exemplary method 200 of operating a wearable sensor system 104 to evaluate a drug or treatment of interest by monitoring the progression of a disease, injury, or condition in a subject (e.g., to identify or confirm a therapeutic agent or an effective dosage thereof), according to an illustrative embodiment. The method 200 includes placing (202) a wearable device (e.g., 104) including a wearable printed sensor (e.g., 110) on an animal model (e.g., 102), e.g., on the skin. The method 200 then includes administering (204) a test agent or introducing a test stimulus to the animal model (e.g., 102).

[0062] In another embodiment, the therapeutic agent is capable of ameliorating damage in a subject.

[0063] In some embodiments, the subject has been administered an agent of interest. In some embodiments, the subject has been subjected to injury. In some embodiments, the subject has been administered an agent of interest and subjected to injury.

[0064] It should be understood that the term "injury" refers to the injury to the subject's body caused by external force.Injury may include, but is not limited to, wound, head injury, penetrating head injury, closed head injury, muscle injury, masseter muscle injury, brain injury, acquired brain injury, direct hit contralateral injury, diffuse axonal injury, frontal lobe injury, nerve injury, spinal cord injury, brachial plexus injury, sciatic nerve injury, axillary nerve injury, soft tissue injury, tracheobronchial injury, acute kidney injury, anterior cruciate ligament injury, musculoskeletal injury, articular cartilage injury, acute lung injury, pancreatic injury, thoracic aorta injury, bile duct injury, knee injury, medial knee injury, hand injury, chest injury.In some embodiments, injury can include induced masseter muscle injury.

[0065] A wound is an injury in which the skin is torn, cut, or punctured (open wound) or where blunt trauma causes bruising (closed wound). A wound can be defined as any damaged area of ​​tissue that may or may not produce fluid. In addition, wounds or ulcers can result from traumatic or pathogenic disruption of epithelial layers, such as the gastrointestinal, renal, urethral, ​​or ureteral epithelium, or disruption of endothelial layers, such as the vascular or cardiac endothelium. Examples of such wounds include, but are not limited to, abdominal wounds or other large or incisional wounds as a result of surgery, trauma, sternotomy, fasciotomy, or other conditions, disconnected wounds, acute wounds, chronic wounds, subacute and dehiscence wounds, traumatic wounds, vascular wounds (e.g., venous ulcers, arterial ulcers), skin flaps and skin grafts, surgical wounds, lacerations, abrasions, contusions, hematomas, burns, diabetic ulcers, pressure ulcers, stomas, cosmetic wounds, traumatic ulcers, neuropathic ulcers, venous ulcers, arterial ulcers, chronic wounds, non-healing wounds, or any combination thereof. Wounds may include easily accessible or difficult to access wounds, exposed and hidden wounds, large and small wounds, regular and irregular shaped wounds, and planar and topographically irregular, non-uniform, or complex wounds. The wound may be present at a site selected from the trunk, extremities, and extremities, such as the heel, sacrum, shaft, groin, shoulder, neck, leg, foot, finger, knee, armpit, arm, and forearm, elbow, hand, or any combination thereof. In some embodiments, the wound may be a vascular wound. In some embodiments, the wound may be a surgical wound. In some embodiments, the wound may be a venous ulcer. In some embodiments, the wound may be an arterial ulcer. In some embodiments, the wound may be present on the extremities and extremities. In some embodiments, the wound may be a non-healing wound. A progressive wound refers to a wound that does not progress in healing in a timely manner, usually within a period of 4 weeks to 3 months (e.g., 1 month to 2 months, 2 months to 3 months, or 1.5 months to 2.5 months). In some embodiments, the wound may exhibit delayed healing. For example, the wound does not progress in healing in a timely manner, usually within a time frame of 4 weeks to 3 months (eg, 1 month to 2 months, 2 months to 3 months, or 1.5 months to 2.5 months).

[0066] In some embodiments, the method may be a method of monitoring volumetric muscle loss (VML), the method can include acquiring EMG signals from a wearable device placed over a target muscle of a subject, and using the acquired EMG signals to assess a VML injured masseter muscle and provide real-time continuous monitoring of the VML.

[0067] The method 200 then includes acquiring (206) a biophysical signal (e.g., 108) continuously (212) from a wearable device (e.g., 104) comprising a skin-mountable printed sensor on the skin of the subject. The method 200 includes storing (208) the biophysical signal (e.g., via data acquisition system 128).

[0068] Next, the method 200 includes analyzing (210) the acquired signals by using the acquired signals to assess disease progression in the subject, injury to the subject, or any combination thereof, so as to provide real-time, continuous monitoring of an electrophysiological parameter of the subject.

[0069] In some embodiments, administration of a test agent or test stimulus (204) to an animal model (e.g., 102) may be performed once or multiple times over the course of a study. In the example of FIG. 2B, administration of a test agent or test stimulus 204 (shown as 204a) to an animal model (e.g., 102) is shown to be performed once. A wearable device (e.g., 104), for example a skin-wearable printed sensor, can then continuously acquire (212) a biophysical signal (e.g., 108). In some embodiments, the period of data acquisition is greater than one week or month.

[0070] In another example of Figure 2B, administration 204 (shown as 204a, 204b, 204c, 204d) of a test agent or test stimulus to an animal model (e.g., 102) is shown to occur multiple times over the course of a study. The wearable device (e.g., 104) can acquire (206) a biophysical signal (e.g., 108) continuously (212). In some embodiments, the period of data acquisition is greater than one week or month.

[0071] The skin-wearable printed sensor may include one or more stretchable graphene sensors configured to obtain, for example, an electrocardiogram (ECG) sensor, an electroencephalogram (EEC) sensor, an electromyogram (EMG) sensor, or any combination thereof, as part of, for example, an electrical sensor, an impedance sensor, an infrared sensor, or any combination thereof. The skin-wearable printed sensor may include at least two electrodes, a conductive flexible film, and an elastomeric substrate. In some embodiments, the polymer layer may include polyimide (PI).

[0072] 3A, 3B, 3C, and 3D each show the example skin-wearable sensor system 104 (shown as 104a) of FIG. 1, according to an illustrative embodiment.

[0073] In the example of FIG. 3A, the sensor system 104a includes a multi-layer flexible circuit 302 (shown as 302a, 302b, 302c) with an electrode array 110 (shown as 110b) fabricated on the underside of a bottom layer 302c. In some embodiments, the PI-Cu-PI-Cu-PI multi-layer 302 can be laminated on a polydimethylsiloxane (PDMS) coated 4-inch wafer (not shown - see FIG. 3). The fabricated circuit and electrodes can be removed from the carrier substrate and transferred to a soft silicone elastomer 304. A functional microchip 306 (e.g., a processor, analog-to-digital converter, networking chipset, among others) can be soldered to an exposed pad on the circuit and covered with an elastomer 308. A rechargeable lithium battery (not shown) can be attached to the circuit, and the electrodes and circuit can be connected with a flexible conductive film.

[0074] In the example of Figure 3B, the sensor system 104B includes the multi-layer flexible circuit 302 (shown as 302a, 302b, 302c) of Figure 3A. The multi-layer flexible circuit 302 includes a stretchable pad 110 (shown as 110c) fabricated on the underside of the bottom layer 302c. The sensor system 104b further includes an LED and photodiode circuit 310 attached to the stretchable pad 110c, or other integrated sensor as described herein.

[0075] In the example of Figures 3C and 3D, the sensor system 104 (shown as 104c and 104d, respectively) includes a main circuit 312 comprising the multi-layer flexible circuit 302 (shown as 302a, 302b, 302c) of Figure 3A. The main circuit 312 includes the external sensors 122 (shown as 122a, 122b, respectively) and is connected to them via the flexible circuit 314 through a connector 316. In the example shown in Figure 3C, the external sensor 122a includes an electrode array (e.g., a stretchable electrode array). In the example shown in Figure 3D, the external sensor 122b includes a stretchable pad 110 (shown as 110d) fabricated under the bottom layer of the flexible circuit membrane 314 (shown as 314a). The sensor system 104b further includes an LED and photodiode circuit 310, or other integrated sensor as described herein.

[0076] Exemplary Circuit FIG. 4 shows an example circuit and layout of a sensor system (e.g., 104, 104a, 104b, 104c, 104d). In the example shown in FIG. 4, the sensor system includes printed stretchable electrodes fabricated on a soft elastomeric substrate. The circuit is designed to be stretchable over a small dimension (6 cm 2 ) and thickness (less than 2 mm) and is lightweight (1.63 g). After integrating a rechargeable battery (40 mAh capacity) with a slide switch, the total weight of the circuit is 3.17 g. The small battery allows the active wireless system to continuously record multiple signals for 6 hours.

[0077] The circuit design 400 includes integrated functional components, including a Bluetooth microprocessor capable of delivering measured sensor and motion signals to a portable device. Received signal strength indication (RSSI) can provide a wireless communication range of 5m with a sustained data transmission rate of 1104 bytes / sec. The transmitted sensor and motion signals can be displayed and stored on the portable device with a customized app. The soft and flexible circuit exhibits mechanical reliability even under complete folding (180 degrees with a 1.5mm radius of curvature) during cyclic loading (100 cycles).

[0078] 4, the circuit 400 includes a sensor IC 402, an antenna network 404, a Bluetooth and microprocessor 406, a voltage regulator 408, a battery charging IC 410, and a motion sensor IC 412. Table 414 provides a description of the specific components. Exemplary values ​​for the components include 0.1 μF (c1, c2, c10, c12, c14, c19, c22, c26), 0.1 nF (c20), 1 nF (c8, c9), 1 μF (c7, c18, c23), 10 uF (c3, c4, c5, c6, c11, c13), 1 pF (c27), 15 pF (c15), 4.7 μF (c16, c17, c21), 12 pF (c24, c25, c28, c29), 30 kΩ (R1, R2), 1 MΩ (R3, R4), 2 kΩ (R5), 100 kΩ (R6), 2.2 uH (L1), 2.7 nH (L2), and 3.9 nH (L3).

[0079] An exemplary external sensor includes a skin-mountable electrode fabricated using a nanofabrication process. For example, the sensor may include two or more electrodes fabricated by aerosol jet printing (AJP) in a serpentine shape to provide stretchability. A conductive flexible film may be used to connect between the sensor and the soft circuit. The width of the printed graphene membrane may be 0.55 mm. The printed graphene and PI membranes may be laminated onto an elastomer, and the graphene sheets may form a complete film without boundaries for enhanced electrical conductivity. When a thin graphene membrane and PI layer are inserted under the graphene, the electrode can withstand mechanical deformation during the fabrication and measurement process. Figures 5A and 5B show an exemplary fabrication process for a sensor electrode.

[0080] Build details for wearable electronics systems Graphene ink preparation. For electrochemical exfoliation, 10 V can be applied between graphite (Alfa Aesar) and Pt foil in an electrolyte solution of ammonium sulfate ((NH4)2SO4, Sigma-Aldrich). The exfoliated graphene can be purified using deionized water (DI water) and the wet powder can be further filtered under vacuum to remove residues. The filtered wet powder of graphene can be dispersed in DI water and concentrated at 15%.

[0081] Table 1 shows exemplary ink and printing parameters for PI and graphene. The resistivity and skin contact impedance of the printed graphene are approximately 2×10 -3 Ωcm and 210.5 kΩ. [Table 1]

[0082] Graphene electrode printing FIG. 5B illustrates a printing process 500 for graphene electrodes. In the example shown in FIG. 5B, the process 500 includes spin-coating (502) PMMA (e.g., 950 PMMA, Kayaku Advanced Materials) on glass at 1000 RPM for 30 seconds and baking at 200° C. for 2 minutes. The process 500 then includes spraying (504) polyimide (PI) ink (PI-2545, MicroSystems) dissolved in N-methyl-2-pyrrolidone (NMP, Sigma-Aldrich) at a ratio of 4:1 with the air atomizer of an aerosol jet printer, depositing using a 300 μm diameter nozzle, and then curing at 250° C. for 1 hour. The process 500 then includes printing (506) 1% of the graphene ink dissolved in NMP using a 200 μm diameter nozzle. The process 500 then includes dissolving the printed electrode in acetone. Next, the process 500 involves peeling 508 the printed graphene layer from the PMMA / glass slide with a water-soluble tape (ASWT-2, Aquasol) and placing it on a silicone elastomer (1 mm thick, 1:2 mixture of Ecoflex 00-30 and gel, Smooth-On) and rinsing the tape with deionized water.

[0083] Making of circuits. Figure 5B also illustrates a microfabrication process 510 for thin film-based circuits. In the example shown in Figure 5B, the process 510 includes spin-coating PDMS (4:1 base-curing ratio) onto a Si wafer at 4000 RPM for 30 seconds (512). A first PI layer (PI-2610, MicroSystems) can be spin-coated at 2000 RPM for 60 seconds, followed by a soft bake at 100°C for 5 minutes and a hard bake at 250°C for 1 hour. The process 510 then includes depositing 0.5 μm thick Cu by sputtering (514).

[0084] The process 510 then includes spin-coating 516 photoresist (PR, Microposit SC1813, MicroChem) at 3000 RPM for 30 seconds. The workpiece can then be aligned with a photomask, exposed to UV light, and developed with a developer.

[0085] The process 510 then includes etching 518 with a Cu etchant (APS-100, Transene). A second PI layer (PI-2545) can be spin coated at 2000 RPM for 60 seconds and soft baked at 100° C. for 5 minutes. It can then be hard baked in a vacuum oven at 240° C. for 1 hour.

[0086] The process 510 then involves spin-coating 520 PR (AZ P4620, Integrated Micro Materials) at 2000 RPM for 30 seconds and soft-baking at 90° C. for 4 minutes. Photolithography is then performed at 15 mJ / cm 2 The workpiece can be exposed to UV light with an intensity of 1000 nm for 100 seconds. The workpiece can then be developed with developer (AZ-400K, Integrated Micro Materials) diluted with DI water (AZ-400K:DI water=1:4). The via holes can be etched using a reactive ion etcher (RIE). A second Cu layer with a thickness of 2 μm can be deposited by sputtering. The process can then be spin-coated with PR (AZ P4620) at 1500 RPM for 30 seconds and soft-baked at 90 °C for 4 minutes. Photolithography can be performed to obtain a 15 mJ / cm 2 The film can be exposed to UV light having an intensity of 1000 nm for 120 seconds and developed. The process can be followed by etching the exposed Cu using a Cu etchant.

[0087] The process 510 then includes spin-coating (522) a third PI layer (PI-2610) at 3000 RPM for 60 seconds, soft-baking at 100° C. for 5 minutes, and hard-baking in a vacuum oven at 240° C. for 1 hour. The process can spin-coat PR (AZP4620) at 900 RPM for 30 seconds and soft-baking at 90° C. for 4 minutes. Photolithography can be performed to expose UV light and develop the PR. The process can etch the exposed PI using RIE.

[0088] The process 510 then includes transferring the microfabricated circuitry to the elastomer by peeling it from the PDMS / Si wafer with water-soluble tape (526). The process 510 then includes attaching the microchip components using screen-printed low-temperature solder paste (528). The process 510 then includes encapsulating the microchip components with the elastomer. Working Example

[0089] Exemplary systems and methods are disclosed, including a craniofacial volumetric muscle loss (VML) developed using a live mouse model and a wireless nanomembrane non-invasive system that integrates skin-mountable printed sensors and electronics for real-time continuous monitoring of VML. A craniofacial VML model using biopsy punch-induced masseter muscle injury shows impaired muscle regeneration and imbalance in muscle-resident stem cell activity. A wearable nanomembrane system with stretchable graphene sensors that can be laminated to the skin over the target muscle is utilized to measure the electrophysiology of active mouse small round masseter muscle during chewing.

[0090] The thin-film electronics can be seamlessly attached to the back of the mouse to allow its natural behavior while providing long-range wireless recording of muscle activity. The noninvasive system provides highly sensitive electromyographic detection for the masseter muscle with and without VML injury. Furthermore, it is demonstrated that the wireless sensor can monitor the recovery after implantation surgery of craniofacial VML. The functional recovery after surgery is shown to be comparable between limb and masseter muscle transplants for treating craniofacial VML. Overall, the presented extensive studies of stem cell biology, nanofabrication, electrophysiology, and signal processing show the enormous potential of our masseter VML injury model and wearable electronic assessment tools for both mechanistic studies and therapeutic development of craniofacial VML.

[0091] Example 1: Real-time functional assessment of craniofacial VML in mice using wireless nanomembrane electronics Figures 6A-6E provide an overview of real-time functional assessment of craniofacial VML in mice using wireless nanomembrane electronics.

[0092] Specifically, FIG. 6A shows a schematic illustration of a wireless electronic system on mouse skin for functional quantification of the masseter craniofacial VML. The EMG of the mouse cheek region is continuously monitored on a portable device. The inset shows the process of punch-induced VML of the masseter muscle and transplantation to treat the masseter VML. The masseter muscle was injured by a biopsy punch as a model of the craniofacial VML. The VML-injured masseter muscle is transplanted with a biopsy piece of the tibialis anterior (TA) or masseter muscle to fill the damaged area. The device includes non-invasive, stretchable electrodes for direct application to the mouse cheek skin and a miniaturized, wireless, soft circuit for attachment to the back of the mouse. During chewing, EMG data is measured by the electrodes on the mouse cheek skin and delivered to a microprocessor in the circuit for data processing. The signal is then wirelessly transmitted to an external portable device for real-time continuous data monitoring and storage. To establish a VML in the masseter muscle, a normal masseter muscle is injured with a 3 mm diameter biopsy punch. The injured muscle area is grafted with a 3 mm biopsy of tibialis anterior (TA) or masseter muscle (Figure 6A, inset).

[0093] FIG. 6B shows a schematic illustration of a multilayer structure of flexible circuits (left) and stretchable sensors (right) on a soft elastomeric substrate, enabling conformal deposition on mouse skin.

[0094] Figure 6C shows a muscle biopsy to generate a VML in the masseter muscle. The middle of the masseter muscle is biopsied with a 3 mm biopsy punch. The arrow indicates the muscle piece from the biopsy. Figure 6D shows an optical image of an active mouse with the device during chewing. Figure 6C shows the muscle biopsy used to generate a masseter VML, while Figure 6D shows that the movement remains unobstructed during monitoring.

[0095] FIG. 6E shows a flow chart for capturing quantitative measurements to analyze muscle function. The flow chart shown in FIG. 6E shows the quantitative measurements for wireless EMG measurement and muscle function analysis. This wearable electronic system can monitor EMG activity in each of the three different craniofacial muscle conditions (normal, VML, VML treated by implantation) in our rodent model. The motion sensor package can also use sensitive accelerometers and gyroscopes to distinguish mouse movements to differentiate chewing movements. The handheld device is embedded with a custom designed app to provide real-time signal display and data storage to analyze muscle function.

[0096] Example 2. Characterization of graphene film electrodes and flexible wireless circuits To quantify muscle function in our craniofacial VML model, EMG signals were monitored using a wearable wireless system, e.g., as described in relation to Fig. 4. Conventional EMG systems in animal studies [35, 38, 39] use bulky and invasive needle-type wired electrodes that penetrate the target muscles. The main problem with these systems is that they are not suitable for use in small mice with active movements. In contrast, this study developed a non-invasive, miniaturized, soft electronic system to provide wireless high-fidelity recording of muscle function with naturally moving mice.

[0097] The biocompatibility of the sensor attached to the tissue is a feature that can be used to provide a safe and continuous use with no side effects. [40, 41] In addition, the cytotoxicity of the electrodes may damage the skin cells in the VML injury area when measuring muscle activity. Characterization of the biocompatibility of the printed graphene electrodes was performed with human keratinocyte cells. The number of live cells on the graphene and the control (polystyrene cell culture dish) was determined by fluorescence intensity.

[0098] Fluorescence images were taken of keratinocyte cells cultured on two types of substrates, including a control (polystyrene Petri dish, left) and graphene integrated on an elastomer (right). Tests were performed using human primary keratinocyte cells cultured in an incubator at 37 °C with 5% CO2. In the incubator, material samples were placed in 24-well plates and incubated at 5000 keratinocytes / cm. 2 After 7 days in the incubator, the keratinocyte cells were washed with phosphate-buffered saline (Fisher Chemical) and stained with 0.1 ml of calcein blue AM (Thermo Fisher) in 0.9 ml of culture medium. The keratinocytes and reagents were kept in the incubator at 37°C for an additional 10 min. The supernatant was then aliquoted into 96-well plates for further biocompatibility.

[0099] Figures 7A-7J show the results of the characterization of the graphene membrane electrodes and wireless flexible circuits. Specifically, Figure 7A shows a photograph of the stretchable printed electrodes on an elastomeric membrane. Figure 7B shows the cross-sectional (left) and top-view (right) SEM images of the multilayer electrodes. Figure 7C shows an AFM image showing the surface roughness of the printed graphene. The average RMS is 72.9 nm, which ensures uniformity in the printed graphene layers. Figure 7D shows a comparison of the cell absorbance (left) and fluorescence (right) of the control and cultured cells on graphene. The data are analyzed with one-way ANOVA (ns = not statistically significant). The error bars represent the standard deviation. Figure 7E shows a graph representing the relative resistance variation (top) following 60% of the tensile strain change (bottom) over 100 cycles. It can be observed that the absorbance (left graph) and fluorescence (right graph) of the cultured cells in Figure 7D indicate that the printed graphene electrodes have a negligible effect on cell viability. FIG. 7E presents the relative resistance variation of the electrode (top) following 60% of the tensile strain change (bottom) over 100 cycles, indicating reliable mechanical performance under strain.

[0100] Figure 7F shows the RSSI response according to Bluetooth communication distance, showing a sustained data transmission rate of 1104 bytes / sec. Figure 7G shows the handheld device application interface displaying real-time continuous motion (accelerometer and gyroscope) and EMG signals from the wearable sensor system. Figure 7H shows the resistance change of the flexible circuit during cyclic loading (100 times 180° bends with a 1.5 mm radius of curvature), showing a small change in resistance.

[0101] Analysis of the received signal strength indication (RSSI) indicates successful wireless communication distance of 5 m with a sustained data transmission rate of 1104 bytes / s (Figure 7F). The transmitted EMG and kinematic signals are displayed and stored on the portable device with a customized app (Figure 7G). The soft and flexible circuit demonstrates mechanical reliability even under complete folding (180 degrees with a radius of curvature of 1.5 mm) during cyclic loading (100 cycles, Figure 7H), consistent with the results from computational modeling data (Figures 11A and 11B). Figures 11A-11B show the results of computational mechanical simulations. Specifically, Figure 11A shows finite element analysis (FEA) results showing the mechanical compliance of the printed electrodes before (left) and after (right) a uniaxial stretch of 60% (right). Figure 11B shows the FEA results of the bendable circuit at 180°, showing no mechanical failure.

[0102] Example 3 Establishment of craniofacial VML and regeneration of defective masseter muscle To establish the craniofacial VML, the masseter muscle was selected by pulling the mandible upwards because it is the key muscle for mastication. The masseter muscle is composed of the superficial and deep masseter muscles. The superficial masseter muscle is a thick, tendon-like part and connects to the zygoma, while the deep masseter muscle is smaller and connects to the mandible

[42] . Figures 8A-8F show the establishment of the craniofacial VML and the test of defective masseter muscle regeneration.

[0103] Specifically, Figure 8A shows an illustrated diagram of the craniofacial VML of the masseter muscle in mice. Figure 8B shows that a 3 mm biopsy punch induces different degrees of muscle loss between 6-month-old males and females. n=4 per gender. Injuries were applied to the superficial masseter muscle using a 3 mm biopsy punch (Figure 8A), which can generate approximately 8.5% and 15% loss of masseter muscle tissue by losing 8.5 ± 2.3 grams and 7.9 ± 1.2 grams of muscle mass from 6-month-old male and female mice, respectively (Figure 8B). Masseter VML injuries using 3 mm biopsies for female mice exhibited similar critical injury size (15% loss) volume compared to mouse limb VML injuries

[16] , but not for male mice, suggesting that 3 mm biopsies on the masseter muscle of male mice could induce functional impairment, and for the remaining experiments, female mice were used. To examine whether muscle regeneration occurs following a 3 mm muscle biopsy-induced injury in the craniofacial VML model, masseter muscles were sectioned at 7 and 28 days after VML injury.

[0104] FIG. 8C shows histology of the masseter muscle 7 days (left) and 28 days (right) after VML injury. Muscle sections were stained with hematoxylin and eosin to visualize nuclei (purple) and cytoplasm (red). The dotted area indicates the non-muscle region. Small square images are magnified in the bottom panel to show cellular elements of the muscle tissue. The arrows in the bottom panel show myofibers containing central nuclei, a characteristic of regenerating muscle fibers. FIG. 8D shows that the non-muscle region occupies the majority of the masseter muscle at 7 and 28 days after injury. Error bars represent standard error of the mean (SEM). FIG. 8C shows histology data of the midpoint of the VML injury of the masseter muscle stained via hematoxylin and eosin staining, which shows that the non-muscle region (dotted line in FIG. 8C) is filled with non-muscle cells, which may be immune cells and fibrosis. The nonmuscle area comprised approximately 40% of the injured masseter muscle at 7 days post injury (dpi) and remained intact at 28 dpi (Figure 8D). Limited muscle regeneration was observed at the edge of the injury area by measuring the cross-sectional area of ​​muscle fibers containing central nuclei, a characteristic of regenerating muscle (arrows in the bottom image of Figure 8C).

[0105] Figure 8E shows that few regenerated muscle fibers were very low at 7 and 28 days after injury. Data are analyzed by Student's t-test. ns = not statistically significant. Figure 8F shows the results of fibrosis in the masseter muscle at 7 days (left) and 28 days (right) after VML injury. Muscle sections were stained with Masson's trichrome stain to visualize fibrotic areas (blue) from muscle tissue (brown). The number of regenerated muscle fibers was about 2% of the total muscle fibers in the masseter muscle at 7 dpi and 28 dpi (Figure 8E). In addition, fibrotic muscle regeneration in the nonmuscle area of ​​the masseter muscle after 7 and 28 days VML injury was detected by Masson's trichrome stain (Figure 8F). Defective muscle regeneration after VML was supported by reduced satellite cells in masseter muscles with VML (Figure 12), whose satellite cells were essential muscle stem cells for muscle regeneration upon injury, compared with those in freeze-induced masseter injury [7]. Figures 12A-12D show the results of filtered EMG signals of mice after VML injury. There is no signal variation of chewing behavior due to masseter muscle loss between Figure 12A (non-fed) and Figure 12B (fed). Chewing EMG signals of three mice after 30 days of injured VML (Figure 12C) and with intact masseter muscle (Figure 12D).

[0106] In addition, fibrotic preadipocytes, muscle mesenchymal stem cells that mediate fibrosis and fat deposition in chronic muscle injury [43, 44], were significantly increased in VML masseter than in freeze-induced masseter injury (Figure 13), which may induce fibrosis in VML masseter. These results demonstrate that craniofacial VML produced by 3 mm biopsy injury results in defective muscle regeneration, similar to that seen in limb VML experiments using the same method.

[16]

[0107] Figures 13A-13E show the results of stem cell dysregulation in the masseter muscle 3 days after VML injury. Specifically, Figure 13A shows the experimental scheme. Tamoxifen induces Pax7 cre / ERTtdTomato mice were injected with tamoxifen for 5 days to induce tdTomato fluorescent expression in satellite cells. VML or freeze injury was performed 10 days after tamoxifen injection. Mononuclear cells were isolated for flow cytometry analysis 3 days after injury. Figure 13B shows a representative dot plot of satellite cells gated by red fluorescent protein (tdTomato) using flow cytometry. Figure 13C shows that some satellite cells from VML-injured masseter muscles are comparable to those in uninjured masseter muscles. Satellite cell numbers are normalized with the average satellite cell number of uninjured muscles. Freeze-injured muscles serve as positive controls. Error bars represent standard deviation of the mean (SEM). Figure 13D shows that surface markers (Cd31 - , CD45 - , Sca1 + Figure 13E shows a representative histogram of fibro-adipocytes (FAPs), defined as fibroblasts (FAPs) with satellite cells (SSCs) in the VML-injured masseter muscle. Data were analyzed by one-way ANOVA and Kruskal-Wallis method for post-hoc comparisons. *p<0.05.

[0108] Example 4 Demonstration of a wireless, wearable EMG system to assess function of the VML-injured masseter muscle Figures 9A-9G show the operation of a wireless wearable EMG system for evaluating the function of VML-injured masseter muscles. Specifically, Figure 9A shows a fully integrated wireless wearable electronics on a thin medical patch for non-invasive diagnosis of VML in mice, for example.

[0109] Figure 9B shows a photograph showing the ultra-thin stretchable EMG sensor attached to the cheek (left) of a nude mouse, and the flexible circuit on the back (right). The device is completely covered with a thin film patch for easy attachment to the skin of the target mouse. The stretchable EMG electrode is attached directly to the mouse's cheek (left image of Figure 9B), while the flexible wireless circuit is attached on the back of the body (right image of Figure 9B).

[0110] Figure 9C shows a photograph of the experimental setup with a mouse in a cage. Real-time continuous movement and EMG data are monitored and recorded by a handheld device with an embedded application. Real-time continuous muscle function is monitored by the wearable device and tablet during the mouse's natural activities, including feeding and walking around in the cage.

[0111] FIG. 9D shows a comparison of real-time EMG signals measured in an uninjured mouse during resting state (top) and chewing phase (bottom), showing clear signal differences. FIG. 9D captures real-time EMG data recorded from a normal mouse during resting state (top) and chewing phase (bottom), displaying increased peak-to-peak voltage. The amplitude of the chewing EMG signal is much smaller compared to the typical intermittent movement artifacts measured by the mouse (FIG. 14). FIG. 14 shows the filtered EMG signal of an uninjured mouse during feeding. The movement artifacts produce significant and distinguishable amplitudes compared to the chewing signal.

[0112] To quantify muscle function after VML injury, the EMG activity of the masseter muscle was monitored 30 days after VML injury. Figure 9E shows photographs of an uninjured mouse (top) and a mouse after VML injury (bottom) after 30 days. The arrows indicate the location of the VML injury area. Figure 9F shows representative RMS EMG signals during chewing corresponding to the two cases in Figure 9E. In addition, both the root mean square (RMS) EMG signals (Figure 9F) and their signal-to-noise ratio (SNR) values ​​(Figure 9G) showed high significance (p-value < 0.01) between the two groups, suggesting that the VML-injured muscle had not recovered even one month after injury, which is consistent with our histological findings in Figure 8. A clear signal difference is observed between the uninjured case (top) and the VML-injured masseter muscle (bottom) at 30 days after injury. FIG. 9G shows the summarized EMG SNR data between the uninjured and VML-injured masseter muscles during chewing. Data were analyzed by unpaired two-tailed Student's t-test. **p<0.01.

[0113] Example 5. Monitoring functional recovery after transplantation of VML-injured masseter muscle Figures 10A-10G show the operation to monitor the functional recovery of VML-injured masseter muscles after transplantation. Specifically, Figure 10A shows the experimental scheme showing biopsy punch-induced VML regions of masseter muscles of immunodeficient mice (NRG) filled with biopsy pieces from TA (blue) or masseter muscles (red) of wild-type mice. EMG measurements and fibrosis analysis have been performed 30 days after VML injury / transplantation. Craniofacial VML was treated in the clinic with autologous limb muscle transplants [45-47]. To evaluate the origin of transplanted muscles for craniofacial VML treatment, transplants of masseter or tibialis anterior (TA) muscles were performed in the VML region of masseter muscles.

[0114] Figure 10B shows that VML injured masseter muscles with TA and masseter muscle grafts are sectioned and labeled with Col VI antibody (red) to measure fibrosis. DAPI staining is used to label nuclei in muscle sections. Masseter or TA muscles were biopsied from wild-type mice using a 3 mm biopsy punch and transplanted into the VML (3 mm biopsy area) to ensure a 1:1 volumetric match of the masseter muscles of severely immunodeficient mice (Figure 10B).

[0115] Figure 10C shows that the mean intensity of Col VI staining indicates that transplantation of TA or masseter muscles produced comparable levels of fibrosis in the transplanted VML-injured masseter muscles, n=3 for each group, and error bars represent standard error of the mean. Data are analyzed using two-way ANOVA. p**<0.01. These mice are used in this transplantation study because they have minimal chance of transplant rejection from the host's immune system.

[48] To ensure proper muscle contractility, whole biopsied masseter or whole TA muscles were positioned to align the fibers between the donor and recipient muscles. EMG activity of the transplanted masseter muscles was measured for 30 days postoperatively. To measure fibrosis, muscle sections from uninjured and VML-injured muscles with TA and masseter muscle transplants were labeled with anti-collagen VI (Col VI) antibody (red) (Figure 10C).

[0116] Figure 10D shows a comparison of filtered EMG of uninjured muscle (top) and VML-injured masseter muscle with transplant of TA muscle (middle) or masseter muscle (bottom). The intensity of Col VI signal was measured to estimate the degree of fibrosis in uninjured muscle (contralateral) and VML-injured masseter muscle with transplant.

[0117] FIG. 10E shows the RMS-EMG signals measured from the TA muscle (left) and the masseter-transplanted masseter muscle (right). Fibrosis levels are significantly higher in the VML with the transplanted masseter muscle compared to the uninjured contralateral masseter muscle. However, transplantation of the TA or masseter muscle does not produce different levels of fibrosis in the transplanted VML injured masseter muscle. EMG activity was also monitored with wearable membrane electronics to identify functional recovery of the masseter muscle after different muscle transplants (FIG. 19). A decrease in EMG response was seen in both transplanted muscles compared to the contralateral muscles during mastication (FIG. 10E).

[0118] FIG. 10F shows that the SNR values ​​are decreased in the VML along with the transplanted muscle group compared to the uninjured muscle (n=3). TA or masseter transplantation results in a similar level of functional recovery to the VML injured masseter. The signal amplitudes of both transplanted muscles appear to be comparable (FIG. 10F). The summarized SNR values ​​in FIG. 10G show that the muscle function of both VML / transplanted masseter muscles is partially restored compared to the uninjured contralateral masseter. In summary, TA or masseter transplantation results in a similar level of fibrosis and functional recovery to the VML injured masseter.

[0119] experiment Mice used in the study: C57BL / 6J mice (Jax000664) (female n=6 and male n=4), Pax7 CreERT2 / CreERT2 Mouse (Jax017763), Rosa tdTomato / tdTomato (tdTomato) (Jax007909), NU / J (Jax002019) (Female n=7), NRG (NOD.Cg-Rag1 tm1Mom Il2rg tm1Wjl / SzJ; Jax007799) (females, n = 6) were purchased from Jackson Laboratories (Bar Harbor, ME; www.jax.org). Mice were 5–6 months old as described in the figure legends. CreERT2 / CreERT2 Male mice were homozygous for Rosa tdTomato / tdTomato (tdTomato) and Pax7 CreERT2 / + ;Rosa tdTomato / + (Pax7CreERT2 We obtained 12 female (n=12) tamoxifen-tdTomato mice. To label satellite cells with red fluorescence (tdTomato), 1 mg tamoxifen (Sigma-Aldrich, St. Louis, MO) per 10 g body weight was injected intraperitoneally once a day for 5 days. Experiments were performed according to the approved guidelines and ethical approval from the Institutional Animal Care and Use Committee of Emory University and in compliance with the National Institutes of Health.

[0120] Muscle tissue injury and preparation for histological analysis: Mice were anesthetized by 2.5% isoflurane inhalation using a nose cone. For analgesia, mice were injected subcutaneously with 0.1 mg / kg buprenorphine SR (3-day sustained release) prior to muscle injury. The target injury area was the upper part of the superficial masseter muscle. For VML injury, the masseter muscle was punched with a 3 mm muscle biopsy punch, which was used to generate a critical size VML in the quadriceps muscle of mice by depressing the biopsy punch until it contacted the mandible

[16] . To avoid bleeding, the injury area was selected to avoid cutting the external carotid artery or retrofacial vein, both of which surround the upper and lower parts of the deep and superficial masseter muscles, respectively. For freeze injury, a dry ice-cooled 4 mm metal probe was placed over the masseter muscle for 5 seconds, as previously described [8]. For transplantation surgery, the masseter muscle of NRG mice (recipient side) was punched with a 3 mm muscle biopsy punch. The biopsy area was then filled with biopsied sections of TA or masseter muscle from C57BL / 6 mice (donor side). The masses of the biopsied sections of TA and MASTER muscles were equivalent. After injury or surgery, the skin was closed using absorbable sutures. Animals were euthanized by overdose of isoflurane at the indicated time points. Shallow masseter muscle tissue was dissected, frozen in tissue freezing medium (Triangle Biomedical Sciences), and stored at -80°C. 10 μm thick tissue cross sections were collected every 200 μm using a Leica CM1850 cryostat. To observe muscle tissue, muscle sections were stained with hematoxylin and eosin (H&E) according to the manufacturer's instructions, imaged with an EchoRevolve widefield microscope, and analyzed using ImageJ. To detect fibrosis in muscle sections, slides were stained using Massion's Trichrome Stain Kit (Thermo Scientific) according to the manufacturer's instructions (Advanced Microwave Staining Protocol). Slides were rehydrated in PBS before staining and imaged using an EchoRevolve widefield microscope.To measure fibrosis in muscle tissue, muscle sections were immunostained with anti-collagen VI antibody (Fitzgerald Industries International, 70R-CR009X, 1:300) and AF594-conjugated donkey anti-rabbit antibody for visualization. 4',6-diamidino-2-phenylindole (DAPI) was used for nuclear staining.

[0121] Flow cytometry for cell analysis: To analyze the number of satellite cells and fibroadipose precursors (FAPs) in injured muscles, muscles were dissected and digested with dispase II and collagenase II as previously described.

[53] Isolated mononuclear cells were immunostained with the following antibodies: 1:400 CD45-PE (clone 30-F11; BD Biosciences), 1:4000 Sca-1-PE-Cy7 (clone D7, BD Biosciences), 1:400 CD31-PE (clone 390; eBiosciences). Fibroadipose precursors were counted using the following criteria: CD31 - / CD45 - Sca1 + and satellite cells were analyzed by tdTomato using a BD LSRII cytometric analyzer. + Count by and analyze using FCS Expression 6 Flow Software 6.01.

[0122] Fabrication of nanomembrane electronic systems: The integration of the soft platform with microfabrication techniques enabled integrated wireless portable electronic devices. Device fabrication utilized multiple nanofabrication techniques, including high-resolution printing processes for graphene membrane electrodes [52, 54] and conventional photolithography, metallization processes for thin-film-based circuits [55, 56]. For electrode fabrication, PI and graphene films were printed sequentially as a meandering pattern shape via AJP (AerosolJet200, Optomec) on polymethylmethacrylate (PMMA)-coated glass slides. For circuit construction, PI-Cu-PI-Cu-PI multilayers were laminated on polydimethylsiloxane (PDMS)-coated 4-inch wafers. The fabricated circuits and electrodes were retrieved from the carrier substrate and transferred to a soft silicone elastomer (a 1:1 mixture of Ecoflex 00-30 and Gels, Smooth-On). Functional microchips were soldered onto the exposed Cu pads on the circuits and covered with the elastomer. A rechargeable LiPo battery (40 mAh, Adafruit) was integrated into the circuit. The electrodes and the circuit were connected with a flexible conductive film. A medical film (Tegaderm, 3M) was utilized to cover the device and secure it to the mouse skin as well as prevent external damage. An exemplary description of the fabrication process is provided in conjunction with Figures 5A and 5B.

[0123] Preparation of mice for use with wearable electronics: To acclimate the mice to wearing the devices, mice were placed with a dummy circuit on their backs for 2-4 hours one day prior to the experiment. Food and water were excluded for 18 hours prior to the experiment to stimulate food intake. On the day of the experiment, mice were anesthetized with 2.5% isoflurane inhalation using a nose cone. If necessary, hair from the cheeks and dorsal area was removed with hair removal lotion and wiped with an alcohol pad to ensure that the membrane sensor or device was aligned with the skin. After wearing the sensors and devices, mice were allowed to recover on a heating pad. Mice were offered three food pellets when they were active. Feeding activity was recorded as a reference for the EMG signal.

[0124] Signal processing and EMG signal quantification: Masseter EMG activity during chewing was selected for analysis. EMG activity with high movement signals was excluded from movement artifacts. Raw EMG signals were filtered by a second-order Butterworth band-pass filter with a cutoff frequency of 10–30 Hz. The filtered EMG was converted to an RMS signal to determine peak amplitude and noise. SNR was calculated as follows: [52, 57]

number

[0125] In the formula, A signal is the RMS EMG amplitude during chewing, and A noise is the amplitude during non-feeding. SNRs were collected five times and averaged for analysis.

[0126] Statistical analysis: Statistical analysis was performed using Prism 8.0. Results are expressed as mean ± SEM. Experiments were repeated at least three times unless a different number of repeats is stated in the legend. Statistical tests were performed using unpaired t-tests (Welch's t-test) when two groups were compared, one-way ANOVA and Kruskal-Wallis method for post-hoc comparisons when more than two groups were compared, or two-way ANOVA when samples with two independent variables were compared, as described in the figure legends. p<0.05 was considered statistically significant. Statistical methods, p-values, and sample numbers are indicated in the figure legends. A power analysis of the animal studies was performed (Table 2). [Table 2]

[0127] Consideration The craniofacial region contains approximately 60 muscles that are essential for daily life functions, including eye movement, food intake, respiration, and facial expressions.[1, 2] Although the muscles of the head and limbs are comparable contractile organs, the muscles of the head have some unique characteristics compared to the muscles of the limbs, including a unique embryonic origin[1, 3-5] and differential susceptibility to different types of muscular dystrophies.[6] Even though skeletal muscles can regenerate damaged muscles through the activation of muscle-specific stem cells called satellite cells,[7] regenerative capacity varies among muscles. For example, the masseter muscle, which is important for chewing, has a lower regenerative capacity than the tibialis anterior (TA) muscle[8] because the masseter muscle has fewer satellite cells that show delayed differentiation compared to satellite cells in the limb muscles.[9] In contrast, satellite cells in other craniofacial muscles, such as extraocular muscles, show increased regenerative capacity compared to limb satellite cells.[10, 11] Therefore, understanding the unique characteristics of specific muscles may lead to the development of targeted therapeutic approaches for the treatment of muscle injuries.

[0128] Volumetric muscle loss (VML) refers to the traumatic or surgical loss of skeletal muscle tissue, leading to chronic muscle weakness and impaired muscle function.

[12] VML is often associated with civilian vehicle accident or gunfire injuries as well as military casualties. VML is a clinically challenging problem because it requires surgical autologous muscle transplantation, which causes significant donor site morbidity.

[13] Thus, many research groups have focused on muscle regeneration using myogenic cell therapy and extracellular matrix development in animal limb VML models.[14-17] Among injury-induced VML, craniofacial injuries with soft tissue penetration account for a large proportion of battlefield injuries

[18] and civilian trauma injuries.

[19] Craniofacial VML causes loss of muscle function and severe cosmetic deformity, which can lead to social isolation and psychological inhibition. [19, 20] Several studies have investigated VML in sheet-like muscles similar to the structure of craniofacial muscles, including rat abdominal muscles [21-23] and rat latissimus dorsi. [20, 24] Studies on VML have been performed on craniofacial muscles of larger animals, such as the sheep zygomaticus, highlighting the pathophysiological differences between limb and craniofacial VML.

[25] However, a craniofacial VML mouse model using actual craniofacial muscles has not yet been reported due to the small size of mouse craniofacial muscles. A potential challenge in developing a craniofacial VML mouse model is the lack of functional assessment tools that can monitor the regeneration and recovery of damaged craniofacial muscles in active mice in a noninvasive manner. Current existing electromyography (EMG) systems have limitations in longitudinal studies using mouse models due to the bulky systems that require invasive metal sensors, wires, and multiple electronic components. [26-28] Recent advances in wearable electronics have enabled wireless monitoring of various physiological signals that can be measured on the skin.[29-31] Compact device integration on a soft elastomer platform can provide comfortable wearability without the motion artifacts caused by cumbersome wires and rigid systems.[32, 33] The use of non-invasive and ergonomic factors in the monitoring system / device prevents restriction of movement during measurements and thus allows monitoring physiological responses in a natural ambulatory environment.

[0129] Here, this report introduces nano-membrane electronics for measuring real-time muscle electromyograms on the skin of mouse masseter muscles with and without biopsy punch-induced VML. It was confirmed that the masseter VML model exhibits regeneration of muscle injury. A wireless and wearable electronic system was used to provide real-time EMG monitoring to measure the function of VML-injured masseter muscles in active mice. The system includes an ultra-thin, low-profile, lightweight, and stretchable membrane sensor based on biocompatible graphene and a thin-film soft circuit for data processing that provides seamless attachment to mouse skin without interfering with natural behavior. In vivo demonstration of EMG recording in mouse masseter muscles validates the functionality of the wearable system, which can clearly distinguish the signal difference between mice with and without craniofacial VML. The use of the wireless, non-invasive soft EMG system in active and moving mice is described. Table 3 highlights the work described herein in comparison with previous reports in terms of electrode type, measurement type, recording system, target muscle, and data recording condition. [26,27,34-38] Additionally, this system monitors functional recovery after transplantation surgery to treat VML. It has been shown that there is increased fibrosis and decreased EMG activity in VML-injured muscles after transplantation, regardless of the source of donor muscle. [Table 3]

[0130] Additional considerations Although the present study achieved meaningful continuous EMG monitoring in mice, movement artifacts affected the signal analysis. Despite the thin and soft membrane electrodes, the size of the device was slightly larger than the target muscle. Also, it was observed that the test mice occasionally tried to scratch the device, which can be resolved by further miniaturizing the circuitry and sensors in the second generation device [49, 50]. In addition, a movement sensor was introduced to filter out EMG signals with high motor activity in order to collect EMG activity during chewing. Machine learning-based algorithms can provide automated signal identification and behavior classification for further study [51, 52]. Because EMG signals may differ slightly depending on the area where the electrodes were attached, certain localization with an increasing number of specimens was necessary to obtain accurate results. Nevertheless, the newly developed wireless EMG system showed sufficient sensitivity to determine the function of the masseter muscle. Implantation experiments were performed to verify the effectiveness of craniofacial reconstruction surgery with autologous limb muscle grafts [45-47] using the EMG sensor. Although better outcomes (such as higher EMG) may be obtained when the masseter VML is transplanted with the original muscle type (masseter type I) and masseter due to the recovery of resident stem cells, veins, and nerves, the results are comparable when the masseter VML is transplanted with the appendicular muscle (TA, the majority of the muscle is type IIa / b). However, fibrotic tissue around the transplanted muscle tissue or cellulose tissue in the skin incision area is a collection of accurate EMG measurements. This result again emphasized the need for accurate sensor localization and increased sample number to obtain more accurate results. Overall, the device produced statistically different signals to distinguish between normal, VML-injured, and transplanted VML-injured masseter muscles.

[0131] Although exemplary embodiments of the present disclosure have been described in detail herein in certain instances, it should be understood that other embodiments are contemplated. Thus, the present disclosure is not intended to be limited in scope to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or carried out in various ways.

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Claims

1. 1. A system comprising: a skin-mountable printed sensor; and electronics coupled to the skin-wearable printed sensor.

2. 10. The system of claim 1 , wherein the skin-wearable printed sensor comprises one or more stretchable graphene sensors.

3. 10. The system of claim 1, wherein the skin-wearable printed sensor comprises at least two electrodes, a conductive flexible film, and an elastomeric substrate.

4. 4. The system of claim 3, wherein the electrode comprises a graphene layer in contact with a polyimide (PI) layer.

5. The system of claim 4 , wherein the polyimide layer is in contact with the elastomeric substrate.

6. The system of claim 3 , wherein the conductive flexible film connects the skin-mountable printed sensor with the electronics.

7. The system of claim 1 , wherein the electronics comprises thin-film components.

8. The system of claim 1 , wherein the electronic device comprises a wireless component.

9. The system of claim 1 , wherein the electronics comprises an antenna, bluetooth, a microprocessor, acquisition electronics, a battery, or any combination thereof.

10. 10. The system of claim 1, wherein the skin-wearable printed sensor is selected from an electrical sensor, an impedance sensor, an infrared sensor, or any combination thereof.

11. The system of claim 10 , wherein the electrical sensor is selected from an electrocardiogram (ECG) sensor, an electroencephalogram (EEC) sensor, an electromyogram (EMG) sensor, or any combination thereof.

12. The system of claim 1 , wherein the skin-wearable printed sensor comprises a skin-wearable printed EMG sensor.

13. An animal model comprising: An animal model comprising an animal subject comprising a wearable device comprising a skin-mountable printed sensor on the skin of the animal subject, the animal subject being administered a drug of interest, subjected to injury, or any combination thereof.

14. The animal model of claim 13 , wherein the injury comprises a biopsy punch induced masseter muscle injury.

15. The animal model of claim 13 , wherein the animal model is a craniofacial VML model.

16. 14. The animal model of claim 13, wherein the skin-wearable printed sensor comprises one or more stretchable graphene sensors.

17. 14. The animal model of claim 13, wherein the skin-mountable printed sensor is selected from an electrical sensor, an impedance sensor, an infrared sensor, or any combination thereof.

18. 20. The animal model of claim 17, wherein the electrical sensor is selected from an electrocardiogram (ECG) sensor, an electroencephalogram (EEC) sensor, an electromyogram (EMG) sensor, or any combination thereof.

19. 14. The animal model of claim 13, wherein the skin-mountable printed sensor comprises a skin-mountable printed EMG sensor.

20. The animal model of claim 13, wherein the wearable device comprises a system according to any one of claims 1 to 12.

21. 1. A method for monitoring an electrophysiological parameter of a subject, the method comprising: acquiring a signal from a wearable device comprising a skin-mountable printed sensor on the skin of a subject; and using the acquired signals to assess disease progression in the subject, injury to the subject, or a combination thereof, to provide real-time, continuous monitoring of the electrophysiological parameter of the subject.

22. 22. The method of claim 21 , wherein the skin-wearable printed sensor comprises one or more stretchable graphene sensors.

23. 22. The method of claim 21 , wherein the skin-wearable printed sensor is selected from an electrical sensor, an impedance sensor, an infrared sensor, or any combination thereof.

24. 24. The method of claim 23, wherein the electrical sensor is selected from an electrocardiogram (ECG) sensor, an electroencephalogram (EEC) sensor, an electromyogram (EMG) sensor, or any combination thereof.

25. 22. The method of claim 21 , wherein the skin-wearable printed sensor comprises a skin-wearable printed EMG sensor.

26. The method of claim 21, wherein the wearable device comprises a system according to any one of claims 1 to 12.

27. The method of claim 21, wherein the subject comprises an animal model according to any one of claims 13 to 19.

28. 22. The method of claim 21, wherein the subject has been administered a drug of interest.

29. 22. The method of claim 21, wherein the subject has been subjected to an injury.

30. 22. The method of claim 21, wherein the subject is administered an agent of interest and subjected to injury.

31. The method of claim 21 , wherein the injury comprises an induced masseter muscle injury.

32. 1. A method for identifying a therapeutic agent, the method comprising: contacting a wearable device comprising a skin-wearable printed sensor with the skin of a subject; obtaining a signal from the wearable device on the skin of the subject; administering to said subject an agent of interest; obtaining a signal from the wearable device on the skin of the subject following administration of the agent of interest; comparing the signal in the subject before and after administration of the agent of interest; and analyzing results from the comparing step to evaluate a physiological parameter of the subject; The method, wherein the physiological parameter provides an indication that the agent of interest is a therapeutic agent.

33. 33. The method of claim 32, wherein the therapeutic agent ameliorates damage on the subject.

34. 33. The method of claim 32, wherein the skin-wearable printed sensor comprises one or more stretchable graphene sensors.

35. 33. The method of claim 32, wherein the skin-wearable printed sensor is selected from an electrical sensor, an impedance sensor, an infrared sensor, or any combination thereof.

36. 36. The method of claim 35, wherein the electrical sensor is selected from an electrocardiogram (ECG) sensor, an electroencephalogram (EEC) sensor, an electromyogram (EMG) sensor, or any combination thereof.

37. 33. The method of claim 32, wherein the skin-wearable printed sensor comprises a skin-wearable printed EMG sensor.

38. The method of claim 32, wherein the wearable device comprises a system according to any one of claims 1 to 12.

39. The method of claim 32, wherein the subject comprises an animal model according to any one of claims 13 to 19.

40. 1. A wearable device comprising:

1. A skin-wearable printed sensor comprising: At least two electrodes; A conductive flexible film; A wearable device comprising a skin-mountable printed sensor comprising an elastomeric substrate.

41. 41. The wearable device of claim 40, wherein the electrode comprises a graphene layer in contact with a polyimide (PI) layer.

42. 41. The wearable device of claim 40, wherein the polyimide layer is in contact with the elastomeric substrate.

43. 41. The wearable device of claim 40, wherein the conductive flexible film connects the wearable device with an electronic device.

44. 44. The wearable device of claim 43, wherein the electronics comprise thin film components.

45. 44. The wearable device of claim 43, wherein the electronics include a wireless component.

46. 45. The wearable device of claim 44, wherein the thin film component comprises an antenna, bluetooth, a microprocessor, acquisition electronics, a battery, or any combination thereof.

47. 41. The wearable device of claim 40, wherein the skin-wearable printed sensor comprises one or more stretchable graphene sensors.

48. 41. The wearable device of claim 40, wherein the skin-wearable printed sensor is selected from an electrical sensor, an impedance sensor, an infrared sensor, or any combination thereof.

49. 49. The wearable device of claim 48, wherein the electrical sensor is selected from an electrocardiogram (ECG) sensor, an electroencephalogram (EEC) sensor, an electromyogram (EMG) sensor, or any combination thereof.

50. 41. The wearable device of claim 40, wherein the skin-wearable printed sensor comprises a skin-wearable printed EMG sensor.

51. 1. A system comprising: a skin-mountable printed EMG sensor; and electronics coupled to the skin-wearable printed EMG sensor.

52. 52. The system of claim 51 , wherein the skin-wearable printed EMG sensor comprises one or more stretchable graphene sensors.

53. 52. The system of claim 51 , wherein the skin-wearable printed EMG sensor comprises at least two electrodes, a conductive flexible film, and an elastomeric substrate.

54. 54. The system of claim 53, wherein the electrode comprises a graphene layer in contact with a polyimide (PI) layer.

55. 55. The system of claim 54, wherein the polyimide layer is in contact with the elastomeric substrate.

56. 54. The system of claim 53, wherein the conductive flexible film connects the skin-mountable printed EMG sensor with the electronics.

57. 52. The system of claim 51, wherein the electronics comprises thin-film components.

58. 52. The system of claim 51, wherein the electronic device comprises a wireless component.

59. 52. The system of claim 51, wherein the electronics comprises an antenna, bluetooth, a microprocessor, acquisition electronics, a battery, or any combination thereof.

60. 1. A craniofacial VML model, comprising: A craniofacial VML model comprising an animal subject that has been subjected to a biopsy punch-induced masseter muscle injury, said animal subject being instrumented with a skin-mountable printed EMG sensor.

61. 61. The craniofacial VML model of claim 60, wherein said craniofacial VML model exhibits impaired muscle regeneration and imbalanced muscle resident stem cell activity.

62. 1. A method for monitoring a VML, the method comprising: acquiring EMG signals from a wearable device placed on a target muscle of a subject; and using the acquired EMG signals to assess a VML injured masseter muscle and provide real-time continuous monitoring of the VML.

63. The method of claim 62, wherein the wearable device comprises a system according to any one of claims 1 to 12, or 51 to 59.

64. 63. The method of claim 62, wherein the subject comprises a craniofacial VML model according to claim 60 or 61.

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