WO2023137142A2 - Adhésif de tissu actif et ses utilisations - Google Patents

Adhésif de tissu actif et ses utilisations Download PDF

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WO2023137142A2
WO2023137142A2 PCT/US2023/010734 US2023010734W WO2023137142A2 WO 2023137142 A2 WO2023137142 A2 WO 2023137142A2 US 2023010734 W US2023010734 W US 2023010734W WO 2023137142 A2 WO2023137142 A2 WO 2023137142A2
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tissue
magenta
muscle
stimulation
actuator
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PCT/US2023/010734
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WO2023137142A3 (fr
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David J. Mooney
Sungmin NAM
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President And Fellows Of Harvard College
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/04—Metals or alloys
    • A61L27/06—Titanium or titanium alloys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61H—PHYSICAL THERAPY APPARATUS, e.g. DEVICES FOR LOCATING OR STIMULATING REFLEX POINTS IN THE BODY; ARTIFICIAL RESPIRATION; MASSAGE; BATHING DEVICES FOR SPECIAL THERAPEUTIC OR HYGIENIC PURPOSES OR SPECIFIC PARTS OF THE BODY
    • A61H23/00—Percussion or vibration massage, e.g. using supersonic vibration; Suction-vibration massage; Massage with moving diaphragms
    • A61H23/02—Percussion or vibration massage, e.g. using supersonic vibration; Suction-vibration massage; Massage with moving diaphragms with electric or magnetic drive
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/08—Muscles; Tendons; Ligaments
    • A61F2002/0894—Muscles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61H—PHYSICAL THERAPY APPARATUS, e.g. DEVICES FOR LOCATING OR STIMULATING REFLEX POINTS IN THE BODY; ARTIFICIAL RESPIRATION; MASSAGE; BATHING DEVICES FOR SPECIAL THERAPEUTIC OR HYGIENIC PURPOSES OR SPECIFIC PARTS OF THE BODY
    • A61H2201/00—Characteristics of apparatus not provided for in the preceding codes
    • A61H2201/16—Physical interface with patient
    • A61H2201/1602—Physical interface with patient kind of interface, e.g. head rest, knee support or lumbar support
    • A61H2201/165—Wearable interfaces
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/16—Materials with shape-memory or superelastic properties
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/30—Materials or treatment for tissue regeneration for muscle reconstruction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/20—Applying electric currents by contact electrodes continuous direct currents
    • A61N1/26—Electromedical brushes; Electromedical massage devices ; Combs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/322—Electromedical brushes, combs, massage devices
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents
    • A61N1/36—Applying electric currents by contact electrodes alternating or intermittent currents for stimulation
    • A61N1/36003—Applying electric currents by contact electrodes alternating or intermittent currents for stimulation of motor muscles, e.g. for walking assistance

Definitions

  • Mechanical stimulation is an attractive approach to promote tissue regeneration and rehabilitation, as it can provide localized activity, may avoid pleiotropic effects, and also lead to a simplified regulatory approval process, as compared to traditional drug therapies. It has been established that cells and tissues can respond to microenvironmental biophysical cues and external mechanical stimuli through a process termed mechanotransduction, which converts physical signals into changes in cell function 1–3 . Studies investigating the impact of mechanical stimulation on cells and engineered tissue constructs have found that mechanical stimuli (e.g., stretching) can regulate cytoskeleton remodeling 4 , cell proliferation 5–8 , force generation 9–11 , and cell differentiation 12 .
  • mechanical stimuli e.g., stretching
  • muscle atrophy results from physical inactivity or denervation, and also occurs as a systematic response to fasting and various diseases, including cancer associated cachexia and diabetes 30–32 .
  • cancer associated cachexia and diabetes 30–32 a chronic myofibrillar and soluble proteins
  • muscle mass is lost due to the accelerated degradation of myofibrillar and soluble proteins and the decreased synthesis of new proteins, resulting in weakened muscle function and increased disability 30 .
  • muscle atrophy can be prevented or slowed through exercise 33 , this is often not feasible (e.g., patients under bed rest).
  • MAGENTA mechanically active gel-elastomer-nitinol tissue adhesive
  • Figure 1a The mechanical actuation of MAGENTA is enabled by a soft robotic actuator made of a shape memory alloy (nitinol) spring and an elastomer.
  • Tissue adhesion is achieved by incorporating a hydrogel- based adhesive, which interfaces the underlying tissue and transmits the actuation to the target tissue, providing mechanical stimulation in a localized and controlled manner.
  • this mechanical stimulation was found to attenuate muscle atrophy as well as promote recovery from atrophy. While this study is mainly focused on using electric voltage to generate the actuation of MAGENTA, we further demonstrate that MAGENTA can also be actuated by laser irradiation, suggesting the possibility of remote control.
  • the present disclosure provides a tissue stimulator comprising: a soft actuator adhered to said tissue; wherein soft actuator comprises a shape memory alloy and an elastomer; wherein the soft actuator is adhered to said tissue via a biocompatible adhesive system; wherein the soft actuator creates a mechanical stimulation to generate tension on said tissue.
  • the shape memory alloy of the tissue stimulator according to the first embodiment is nitinol.
  • the biocompatible adhesive system of the tissue stimulator according to the first or second embodiment is adhered to the soft actuator via a crosslinking agent.
  • the crosslinking agent of the third embodiment is benzophenone.
  • the biocompatible adhesive system of the tissue stimulator according to the first, second, third, or fourth embodiment comprises a hydrogel comprising a first polymer network and a second polymer network, wherein said first polymer network comprises covalent crosslinks and said second polymer network comprises ionic crosslinks.
  • the biocompatible adhesive system of the fifth embodiment further comprises a bridging polymer.
  • the bridging polymer is chitosan.
  • the biocompatible adhesive system of the sixth embodiment further comprises a coupling agent.
  • the first polymer network and the second polymer network are covalently coupled.
  • the first polymer network is acrylamide and the second polymer network is sodium alginate.
  • the hydrogel does not include a magnetic material.
  • the elastomer stiffness of the tissue stimulator of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth embodiment is approximately 130 kPa.
  • the biocompatible adhesive stiffness of the tissue stimulator of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or eleventh embodiment is approximately 60 kPa.
  • mechanical stimulation of the soft actuator according to the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, or twelfth embodiment is created by a stimuli which causes said soft actuator to contract.
  • the stimuli of the thirteenth embodiment is an electrical voltage or laser irradiation.
  • the present disclosure provides a method of preparing a tissue stimulator according to the first embodiment, comprising the steps of: preparing a soft actuator; preparing a biocompatible adhesive system; adhering said biocompatible adhesive system to said soft actuator; and adhering said soft actuator to a tissue.
  • the step of preparing a soft actuator according to the fifteenth embodiment comprises the steps of: stretching a shape memory alloy and holding said stretched shape memory alloy in a mold; adding elastomer material to the mold; and removing the elastomer material and said shape memory alloy from said mold.
  • the step of adhering said biocompatible adhesive system to said soft actuator comprises the steps of: treating a surface of said elastomer material with a crosslinking agent; preparing a hydrogel; adding said hydrogel to said treated surface of said elastomer material; and irradiating said crosslinking agent to crosslink said elastomer material and said hydrogel.
  • the step of adhering said soft actuator to a tissue comprises the steps of: adding a bridging polymer to said hydrogel cross-linked to said elastomer material; and compressing said adhesive polymer and tough hydrogel with said tissue.
  • the present disclosure provides a method of using a tissue stimulator according to the first embodiment, comprising the step of stimulating said tissue stimulator, and wherein said stimulation causes said tissue stimulator to generate a tension on said tissue. In some embodiment the stimulation causes the tissue stimulator to contract.
  • the step of stimulating said tissue stimulator according to the nineteenth embodiment comprises the step of irradiating said tissue stimulator or applying an electrical voltage to said tissue stimulator.
  • Figure la A schematic depicting the potential application of MAGENTA and its actuation principle to generate mechanical stimulation.
  • Figure lb An example of a soft actuator fabricated with a shape-memory alloy (SMA) spring and an elastomer.
  • SMA shape-memory alloy
  • Figure 1c An example of contraction and relaxation of an actuator during actuation. The actuator contracted immediately upon application of voltage, and relaxed once the voltage was discontinued.
  • Figure 2a A schematic illustration of force balance between spring contraction and elastomer resistance during actuation.
  • E20, E50, D10, D20 and D30 represent EcoflexTM 00-20, 00-50, Dragon SkinTM 10, 20 and 30, respectively. Data are shown as mean ⁇ SD.
  • Figure 2d Digital (left) and thermal images of an actuator before (middle) and after (right) application of voltage.
  • Figure 2e Time evolution of temperature averaged along the edge of actuators during cyclic actuation. The frequency of actuation was 0.1 Hz. Each peak indicates when the voltage was applied during the cycle.
  • Figure 2h A simulation model combining the actuator, adhesive and tissue model to predict tissue deformation.
  • Figure 2i Predicted tissue deformation as a function of elastomer stiffness. Black arrow indicates the greatest predicted tissue strain. Data are shown as mean ⁇ SD.
  • Figure 2j Predicted tissue deformation as a function of adhesive gel stiffness. Data are shown as mean ⁇ SD.
  • Figure 2k Predicted tissue deformation as a function of adhesive gel thickness. Data are shown as mean ⁇ SD.
  • Figure 3a A schematic illustration of a robust interface formation between elastomer, tough gel and tissue.
  • Figure 3b Compressive stress of tough hydrogels during cyclic deformation.
  • Figure 3d Photos of T-peeling tests for the elastomer-hydrogel (left) and hydrogel-tissue (right) interface.
  • Figure 3e Curves of the peeling force per width versus displacement for the elastomer-hydrogel and hydrogel-tissue interface.
  • Figure 3g A photo of actuator-adhesive hybrid.
  • Figure 3h Ex vivo application of MAGENTA. Photos of skeletal muscle tissue deformation generated during actuation of MAGENTA (top) and corresponding displacement fields (bottom) acquired by the paint speckle tracking analysis. Scale bars, 1 mm.
  • Figure 3j In vivo application of MAGENTA. Photos of skin surrounding the implanted MAGENTA during actuation.
  • Figure 3k Ultrasound images of tissue deformation during actuation. Scale bars, 1 mm.
  • Figure 31 A kymograph of tissue displacement, based on ultrasound images, during MAGENTA actuation. The specific line tracked over time is indicated in the solid orange line.
  • Figure 3m Estimation of muscle displacement as a function of tissue depth. Data are shown as mean ⁇ SD.
  • Figure 4a Experimental design and timeline of hindlimb immobilization, mechanical stimulation and analysis.
  • Figure 4b Immunoblot of YAP for untreated muscles (Immo) and mechanically stimulated muscles (MS). GAPDH, glyceraldehyde- 3 -phosphate dehydrogenase.
  • Figure 4c Fluorescence images of YAP in muscle tissues treated with and without mechanical stimulation. Yellow arrowheads indicate nuclear localized YAP. Scale bars are 20 pm.
  • Figure 4d Immunoblot of MRTFA for untreated muscles (Immo) and mechanically stimulated muscles (MS).
  • Figure 4e Fluorescence images of MRTFA in muscle tissues treated with and without mechanical stimulation. Yellow arrowheads indicate nuclear localized MRTFA. Scale bars are 20 pm.
  • Figure 4h Immunoblots of puromycin.
  • Figure 4i Immunoblots of MuRFl for muscles with and without mechanical stimulation.
  • Figure 5a Representative images of muscle fibers from mice treated with or without mechanical stimulation after 2 weeks, stained with hematoxylin and eosin (H&E) (top) and laminin (bottom). Scale bars, 100 pm.
  • Figure 5d Photos of lateral gastrocnemius muscle tissue from untreated mice (Immo), mice implanted with MAGENTA but without mechanical stimulation (MAGENTA without MS), and mice implanted with MAGENTA and received mechanical stimulation (MS). Scale bar, 1 mm.
  • Figure 5g Representative muscle force normalized by the muscle weight. Data represent mean ⁇ SD.
  • Figure 6a A schematic illustration of the laser-triggered actuation of MAGENTA.
  • Figure 6b Thermal images of wireless MAGENTA before and after laser irritation.
  • Figure 6c Time evolution of maximum temperature of wireless MAGENTA in response to a laser irradiated at various distances.
  • Figure 6d Actuation of wireless MAGENTA before and after laser irradiation.
  • Figure 6e Ex vivo application of wireless, remote-controllable MAGENTA. Photos of tissue deformation generated during actuation of wireless MAGENTA (top) and corresponding displacement fields (bottom). Scale bars, 1 mm.
  • Figure 6g In vivo application of wireless MAGENTA. Photos of the skin surrounding the implanted wireless MAGENTA before and after laser application. Scale bars, 1 mm.
  • Figure 7a Fabrication process of one embodiment of the soft actuators.
  • Figure 7b Working principle of one embodiment of actuation. Actuation is controlled by electric voltage. Actuation strain is defined as the ratio of the change in spring length to the initial length.
  • Figure 7c Electric circuit to control actuation.
  • Figure 7d Scalable designs of actuators.
  • Figure 8a Compressive stress of elastomers as a function of compressive strain. The neo-Hookean model with incompressibility was used to fit the experimental data and determine the mechanical properties of elastomers.
  • Figure 8b A schematic illustration of the experimental setup to measure the force transmitted to the underlying substrate during actuation.
  • Figure 9a Temperature profile along the axis across the center of the actuator, indicated by the white dotted line.
  • Figure 9b Temperature averaged along the edge of the actuator operating at various frequencies at room temperature. Insets are the voltage signals corresponding to each frequency.
  • Figure 9c Temperature averaged along the edge of the actuator operating at various frequencies at physiological temperature. Insets are the voltage signals corresponding to each frequency.
  • Figure 10a A simulation model for the actuator alone.
  • Figure 10b Strain energies used in the simulation (black). Note that the red data point of the lowest stiffness indicates the same strain energy as the other stiffnesses.
  • Figure lOe Predicted tissue deformation versus tissue stiffness for various elastomer stiffnesses.
  • Figure lOf Predicted tissue deformation versus tissue stiffness for various adhesive gel stiffnesses.
  • Figure 1 la A cartoon depicting application of MAGENTA to phantom tissues (tough hydrogels).
  • Figure 1 lb Top view of phantom tissue deformation during actuation of MAGENTA. Arrows indicate the deformation of the phantom tissue due to the MAGENTA actuation.
  • Figure 11c Side view of phantom tissue deformation during actuation of MAGENTA. Arrows indicate the deformation of the phantom tissue due to the MAGENTA actuation.
  • Figure l id Kymographs of tissue displacement. The specific lines tracked over time are indicated in the yellow lines (left). Scale bar, 1 mm.
  • Figure 12a Photos of actuation at varying actuation cycles.
  • Figure 12b A peeling test after completing the 420 th actuation. Arrows indicate where cohesive adhesion is observed.
  • Figure 13a A schematic illustration demonstrating how the maximum strain during actuation and residual strain after actuation are determined.
  • Figure 13b Examples of actuation strain versus time for actuators with varying elastomer stiffness.
  • Figure 13d Actuation strain of MAGENTA attached to a phantom tissue as a function of time. Actuation strain was normalized to the maximum strain.
  • Figure 13e Actuation strain and relative residual strain of MAGENTA attached to mouse muscle tissue as a function of the number of actuation cycles.
  • Figure 14 Surgical procedure for the application of MAGENTA, which comprises the following steps:
  • Figure 14i First, one hindlimb was fixed with a surgical tape.
  • Figure 14ii The skin around lateral gastrocnemius was incised and gently separated from the muscle to create space for MAGENTA implantation. A small hole was made on the opposite side of the incised skin for the wire to protrude.
  • Figure 14iii One of the electric wires of MAGENTA was first inserted through the hole and bridging polymer was deposited on the tough gel of MAGENTA.
  • Figure 14iv The MAGENTA was then implanted into the muscle.
  • Figure v gentle pressure was applied to the MAGENTA through the skin for 5 minutes.
  • Figure vi The skin incision was closed via suturing.
  • Figure vii The electric wires were trimmed.
  • Figure 15b Cell viability measurements for the cells in Figure 15a. Data are shown as mean ⁇ SD.
  • Figure 15c H&E staining for muscles with and without MAGENTA after 2 weeks of implantation. Scale bars, 100 pm.
  • FIG. 15d Masson’s trichrome staining (MTS) for muscles with and without MAGENTA after 2 weeks of implantation. Scale bars, 100 pm.
  • Figure 16a H&E staining of healthy muscles treated with (Healthy + MS) and without (Healthy) M AGENT A-mediated stimulation. Scale bars, 100 pm.
  • FIG. 16b Masson’s trichrome staining (MTS) of healthy muscles treated with (Healthy + MS) and without (Healthy) MAGENTA-mediated stimulation. Scale bars, 100 pm.
  • Figure 16c Fluorescence images of CD31 staining of healthy muscles treated with (Healthy + MS) and without (Healthy) MAGENTA-mediated stimulation. Scale bars, 100 pm.
  • Figure 17a A schematic illustration of the experimental setup of in vitro cell treatment with MAGENTA.
  • Figure 17b Fluorescence images of myosin heavy chain (MHC) in C2C12 cells treated with (MS) and without (Control) mechanical stimulation by MAGENTA.
  • the fusion index was defined as the percentage of the number of nuclei inside myotubes, indicated by MHC staining, to the total number of nuclei. Scale bar, 50 pm. Stimulation was performed for 2 days with the actuator's maximum strain, frequency of 0.1 Hz, and duration of 5 min per day. Student t tests were used (*P ⁇ 0.05; n.s., not significant). Data are shown as mean ⁇ SD.
  • Figure 18 Spatial distribution of YAP throughout the longitudinal section of stimulated muscle. The Regions of interest are shown as white squares on the left, and magnified images are presented on the right. White arrow heads indicate positive nuclear YAP staining. Scale bars, 100 pm.
  • Figure 19 Spatial distribution of MRTFA throughout the longitudinal section of stimulated muscle. The Regions of interest are shown as white squares on the left, and magnified images are presented on the right. Arrow heads indicate positive nuclear MRTFA staining. Scale bars, 100 pm.
  • Figure 20 Fraction of cells positive for YAP and MRTFA in stimulated muscles as a function of tissue depth. Blue and red dotted lines indicate the averaged values for YAP and MRTFA, respectively, in the various tissue regions indicated in the boxes of Figures 18 and 19. The individual blue circles and red squares represent the average values of YAP and MRTFA nuclear localization, respectively, in the individual tissue regions indicated in the boxes as a function of the distance from the muscle surface to the mid-point of each box. Data were collected from three muscles.
  • Figure 21a Immunoblot analysis of Akt and phosphorylated Akt for muscles with (MS) and without (Immo) stimulation.
  • Figure 21c Fluorescence images of MHC and desmin.
  • Figure 22a Masson’s Trichrome stained sections of muscles treated with (MS) and without (hnmo) stimulation.
  • Figure 22b Quantification of staining intensity, indicative of matrix protein deposition. 15 images in total from 5 muscles were analyzed. Each data point represents each muscle. Scale bar, 100 pm. Student t tests were used, **P ⁇ 0.01. Data are shown as mean ⁇ SD.
  • Figure 23a Timeline of experimental setup for the cessation of mechanical stimulation experiment.
  • Figure 24a Timeline of experimental setup for the long-term mechanical stimulation on disuse muscles experiment.
  • Figure 24b H&E stainingfor untreated (Control) and stimulated (MS) muscles. Scale bar, 100 pm.
  • Figure 24c Masson’s trichrome staining (MTS) for untreated (Control) and stimulated (MS) muscles. Scale bar, 100 pm.
  • Figure 25 a Experimental design and timeline of the recovery experiment.
  • Figure 25d Representative muscle force normalized by the muscle weight.
  • Figure 26a A schematic illustrating the experimental setup for the non- invasive compressive stimulation experiment.
  • Figure 26b Photos of a mouse being treated with compressive stimulation.
  • Figure 26c Experimental design and timeline for the non-invasive compressive stimulation experiment.
  • a compressive force of 0.3 N 0.2 N
  • frequency 1 Hz
  • duty cycle 80% for 5 min per day.
  • data for both the contralateral limb (not immobilized; CONTRA) as well as the immobilized limb are provided. Student t tests were used (***p ⁇ 0.001, ****P ⁇ 0.0001, and n.s., not significant). Data are shown as mean ⁇ SD.
  • data for both the contralateral limb (not immobilized; CONTRA) as well as the immobilized limb are provided. Student t tests were used (***p ⁇ 0.001, ****P ⁇ 0.0001, and n.s., not significant). Data are shown as mean ⁇ SD.
  • a compressive force of 0.3 N 0.2 N
  • frequency 1 Hz
  • duty cycle 80% for 5 min per day.
  • data for both the contralateral limb (not immobilized; CONTRA) as well as the immobilized limb are provided. Student t tests were used (***p ⁇ 0.001, ****P ⁇ 0.0001, and n.s., not significant). Data are shown as mean ⁇ SD.
  • Figure 27a Hyperspectral imaging of shape-memory alloy (SMA). Scale bar, 250 pm.
  • Figure 27b Reflectivity of SMA with respect to varied wavelength.
  • Figure 27d Evolution of actuation strain as a function of time with varied distance.
  • Figure 27f Wireless, remote controllable MAGENTA bonded with tough gel.
  • Figure 28a Schematic illustrations (left insets) and photos of MAGENTA covered with ⁇ 1 mm thick porcine skin along with the underlying muscle tissue.
  • Figure 28b Schematic illustrations (left insets) and photos of MAGENTA covered with ⁇ 1.5 mm thick porcine skin along with the underlying muscle tissue.
  • Figure 28c Schematic illustrations (left insets) and photos of MAGENTA covered with -2 mm thick muscle tissue along with the underlying muscle tissue.
  • Figure 29 An Ashby- style plot of effective strains and tissue stiffnesses for skin, muscle and heart. Effective strain refers to the mechanical strain used in the previous in vitro studies to induce substantial change in cell behaviors. The possible range of mechanical stimulation generated by MAGENTA is indicated in blue. The references to generate the plot are included in Table 1.
  • Figure 30a Uncropped western blot image for Figure 4b.
  • Figure 30b Uncropped western blot image for Figure 4d.
  • Figure 30c Uncropped western blot image for Figure 4h.
  • Figure 30d Uncropped western blot image for Figure 4i.
  • Figure 30e Uncropped western blot image for Figure 21a.
  • soft actuator or “soft robotic actuator” refers to an actuator consisting of elastomeric matrices with embedded flexible materials (e.g., cloth, paper, fiber, particles).
  • Soft actuators for use in the present invention include any actuators known in the art.
  • Exemplary actuators include, but not limited to, a fiber reinforced actuator, a Pneunet bending actuator, a McKibben actuator, a pleated air muscle, a balloon, an inflatable, a motor, a vibrating motor, a cable, an electroactive material, e.g. , a shape memory alloy, an electrostatic or a dielectric elastomer, and combinations thereof.
  • the actuator is fabricated with a shape memory alloy (e.g., nitinol) spring and an elastomer.
  • the controller can also control actuation of the soft actuators by applying an electric signal or by laser irradiation.
  • a shape-memory alloy is an alloy that can be deformed when cold but generally returns to its pre-deformed (“remembered") shape when heated.
  • SMA include, but are not limited to nitinol (nickel titanium), iron-manganese-silicon, copper-zinc- aluminum, and copper- aluminum-nickel based alloys.
  • An elastomer is any rubbery material composed of long chain-like molecules or polymers.
  • elastomer includes, but are not limited to EcoflexTM, Dragon SkinTM, and Polydimethyl siloxane (PDMS).
  • the elastomer has a stiffness of approximately 50 to approximately 250 kPa, in other embodiments the elastomer has a stiffness of approximately 75 to approximately 200 kPa, in other embodiments the elastomer has a stiffness of approximately 100 to approximately 150 kPa, and in some embodiments the elastomer has a stiffness of approximately 130 kPa.
  • the soft actuator is adhered to a tissue using a biocompatible adhesive system.
  • the biocompatible adhesive system has a stiffness of approximately 1 to approximately 100 kPa, in other embodiments the biocompatible adhesive system has a stiffness of approximately 25 to approximately 75 kPa, and in other embodiments the biocompatible adhesive system has a stiffness of approximately 30 to approximately 60 kPa.
  • tissue stimulator refers to a soft actuator adhered to a tissue via a biocompatible adhesive system and which stimulates a tissue when active. In some embodiments the stimulation generates tension on a tissue.
  • the tension applied by the tissue stimulators is generally a contraction tension, or comprises a contracting force on the tissue as opposed to a compressive force. This contracting force or tension is parallel to the surface of the tissue as opposed to perpendicular.
  • the tissue stimulator is activated (i.e. put into the active state, such as generating tension on a tissue) via an external stimulant.
  • the external stimulant is an electrical voltage, and in other embodiments the stimulant is a laser irradiation.
  • tissue has the general meaning of the art. Tissue can refer to an organ, muscle, skin, or other group of cells which function together as a unit.
  • the term “ultrapure” refers to a high purity which is over 60%, 70%, 80%, 90% or more. In some embodiments, “ultrapure” refers to low levels of residual endotoxin, such as below 100 EU/g.
  • the biocompatible adhesive system also referred to as a Tough Adhesive (TA)) herein
  • TA Tough Adhesive
  • the biocompatible adhesive system includes a hydrogel that can be selectively activated with a bridging polymer and an optional coupling agent (e.g., l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and optionally, N-hydroxysuccinimide (NHS)).
  • TA Tough Adhesive
  • alginate-polyacrylamide hydrogels e.g., an alginate-based hydrogel
  • the bridging polymer e.g., chitosan
  • an optional coupling agent e.g., l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)
  • EDC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • the positive charges on the bridging polymer balance the negative charges on the hydrogel, while the coupling agent catalyzes formation of amide bonds between the primary amine polymer and the hydrogel.
  • the activated surface allows the bridging polymer and coupling agent to form chemical bonds to bridge the interface between the hydrogel and the surface of a substrate (e.g., a tissue, a cell, or a device).
  • the term “contacting” is intended to include any form of interaction (e.g., direct or indirect interaction) of a hydrogel and a surface (e.g., tissue or device).
  • Contacting a surface with a composition may be performed either in vivo or in vitro.
  • the surface is contacted with the biocompatible adhesive in vitro and subsequently transferred into a subject in an ex vivo method of administration.
  • Contacting the surface with the biocompatible adhesive in vivo may be done, for example, by injecting the biocompatible adhesive into the surface, or by injecting the biocompatible adhesive into or around the surface.
  • the hydrogel used in the biocompatible adhesive of the invention is an interpenetrating network (IPN) hydrogel.
  • IPPN interpenetrating network
  • an IPN is a polymer comprising two or more networks (e.g., the first polymer network and the second polymer network) which are at least partially interlaced on a molecular scale but not covalently bonded to each other and cannot be separated unless chemical bonds are broken.
  • IPN hydrogels are made by combining covalently crosslinked and ionically crosslinked polymer networks. Alternatively, the first polymer network and the second polymer network are covalently coupled.
  • the first polymer network comprises covalent crosslinks and includes a polymer selected from the group consisting of polyacrylamide (PAAM), poly(hydroxyethy] m ethacryl ate) (PIIEMA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), polyphosphazene, collagen, gelatin, poly (acrylate), poly(methacrylate), poly (methacrylamide), polytacrylic acid), poly(N-isopropylacrylamide) (PNIPAM), poly(N,N-dimentylacrylamide), poly(allylamine) and copolymers thereof.
  • the first polymer network is polyethylene glycol (PEG).
  • the first polymer network is polyacrylamide (PAAM).
  • the second polymer network includes ionic crosslinks and is a polymer selected from the group consisting of alginate (alginic acid or align), pectate (pectinic acid or polygalacturonic acid), carboxymethyl cellulose (CMC or cellulose gum), hyaluronate (hyaluronic acid or hyaluronan), chitosan, K-carrageenan, i-carrageenan and X-carrageenan, wherein the alginate, carboxymethyl cellulose, hyaluronate, chitosan, K-carrageenan, t- carrageenan and k-carrageenan are each optionally oxidized, wherein the alginate, hyaluronate, chitosan, K-carrageenan, i-carrageenan and k-carrageenan optionally include one or more groups selected from the group consisting of methacrylate, acrylate, acrylamide, methacrylamide
  • the second polymer network is alginate, which is comprised of (l-4)-linked b-D-mannuronic acid (M) and a-L-guluronic acid (G) monomers that vary in amount and sequential distribution along the polymer chain.
  • Alginate is also considered a block copolymer, composed of sequential M units (M blocks), regions of sequential G units (G blocks), and regions of alternating M and G units (M-G blocks) that provide the molecule with its unique properties.
  • Alginates have the ability to bind divalent cations such as Ca +2 between the G blocks of adjacent alginate chains, creating ionic interchain bridges between flexible regions of M blocks.
  • the alginate is a mixture of a high molecular weight alginate and a low molecular weight alginate.
  • the ratio of the high molecular weight alginate to the low molecular weight alginate is about 5:1 to about 1:5; about 4:1 to about 1:4; about 3:1 to about 1:3; about 2:1 to about 1:2; or about 1:1.
  • the high molecular weight alginate has a molecular weight from about 100 kDa to about 300kDa, from about 150 kDa to about 250 kDa, or is about 200 kDa.
  • the low molecular weight alginate has a molecular weight from about 1 kDa to about 100 kDa, from about 5 kDa to about 50 kDa, from about 10 kDa to about 30 kDa, or is about 20 kDa.
  • the hydrogel comprises only a single polymer network.
  • the single polymer network comprises the covalently cross-linked polymers disclosed above and in other embodiments the single polymer network comprises the ionically cross-linked polymers disclosed above.
  • the hydrogel used in the present invention does not include a magnetic material .
  • the hydrogels of the invention are highly absorbent and comprise about 30 % to about 98 % water (e.g., about 40%, about, about 50%, about 60 %, about 70%, about 80%, about 90%, about 95%, about 98%, about 40 to about 98 %, about 50 to about 98 %, about 60 to about 98 %, about 70 to about 98 %, about 80 to about 98 %, about 90 to about 98 %, or about 95 to about 98 %water) and possess a degree of flexibility similar to natural tissue, due to their significant water content.
  • the hydrogels of the present invention can be stretched up to 20 times their initial length, e.g., the hydrogels of present invention can be stretched from 2 to 20 times their initial length, 5 to 20 times their initial length, 10 to 20 times their initial length, from 15 to 20 times their initial length, from 2 to 10 times their initial length, from 10 to 15 times their initial length, and from 5 to 15 times their initial length without cracking or tearing.
  • the biocompatible adhesive includes a bridging polymer which is a primary amine polymer. The bridging polymer forms covalent bonds with both the hydrogel and the surface, bridging the two. The primary amine polymer bears positively charged primary amine groups under physiological conditions.
  • the primary amine polymer can be absorbed to a surface (e.g., a tissue, a cell, or a device) via electrostatic interactions, and provide primary amine groups to bind covalently with both carboxylic acid groups in the hydrogel and on the surface. If the surface is permeable, the primary amine polymer can also penetrate into the surface, forming physical entanglements, and then chemically anchor the hydrogel.
  • a surface e.g., a tissue, a cell, or a device
  • the primary amine polymer can also penetrate into the surface, forming physical entanglements, and then chemically anchor the hydrogel.
  • the primary amine polymer includes at least one primary amine per monomer unit.
  • the primary amine polymer is selected from the group consisting of chitosan, gelatin, collagen, polyallylamine, polylysine, and polyethylenimine.
  • polyallylamine (PolyNIh or PAA) is represented by the following structural formula:
  • chitosan is represented by the following structural formula:
  • polyethylenimine (PEI)
  • polylysine is represented by the following structural formula: .
  • Collagen and/or gelatin include approximately -10% amino acid with primary amine (e.g., Arg, Lysine).
  • the biocompatible adhesive also includes a coupling agent.
  • a coupling agent is not required.
  • the coupling agent activates one or more of the primary amines present in the bridging polymer. Once activated with the coupling agent, the primary amine forms an amide bond with the hydrogel and the target surface (e.g., a tissue, an organ, or a medical device).
  • the coupling agent includes a first carboxyl activating agent, wherein the first carboxyl activating agent is a carbodiimide.
  • Exemplary carbodiimides are selected from the group consisting of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, EDAC or EDO), dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC).
  • EDC l-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • DCC dicyclohexylcarbodiimide
  • DIC diisopropylcarbodiimide
  • the first carboxyl activating agent is EDC.
  • the coupling agent further includes a second carboxyl activating agent.
  • second carboxyl activating agents include, but are not limited to, N-hydroxy succinimide (NHS), N-hydroxysulfosuccinimide (sulfo-NHS), hydroxybenzotriazole (HOBt), dimethylaminopyridine (DMAP), Hydroxy-3, 4-dihydro-4- oxo-l,2,3-benzotriazine (HOOBt/HODhbt), l-Hydroxy-7-aza-lH-benzotriazole (HO At), Ethyl 2-cyano-2-(hydroximino)acetate, Benzotriazol- 1 -yloxy-tris(dimethylamino)- phosphonium hexafluorophosphate (BOP), Benzotriazol- 1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate, 7 - Az
  • the biocompatible adhesive system is secured to the soft actuator via a crosslinking agent.
  • the crosslinking agent is activated by UV irradiation, hi other embodiments the soft actuator surface is activated by oxygen plasma treatment followed by the addition of a crosslinking agent.
  • the crosslinking agent is benzophenone.
  • the crosslinking agent is one or more agents selected from 3-(Trimethoxysilyl)propyl methacrylate, (3- Aminopropyl)triethoxy silane, EDC, and NHS.
  • FIG. 7a The fabrication process of the actuator is depicted in Figure 7a. Briefly, a SMA spring (BioMetal, BMX150) was cut into the length of -5.3 mm and connected to sheath- stripped electric wires at both ends. The spring was then stretched to -150% of its initial length and held in a 3D printed mold. The precursors of elastomers (Ecoflex and Dragon skin, Smooth-On) were mixed at 1:1 ratio and poured into the mold. After 4 hours, the elastomer with SMA spring and wires was removed from the mold, thoroughly cleaned with methanol, ethanol and deionized water, and completely dried. Finally, preparation of the actuator was completed by trimming the edges and wires.
  • SMA spring BioMetal, BMX150
  • the size of the actuator can be simply scaled to any dimension, but unless otherwise noted, 8 mm (length) x 1.5 mm (height) x 2 mm (width) was mainly used for this study.
  • stripped electric wires were covered with elastomer, or electric wires sheathed with enamel were used to prevent any electric leakage.
  • the wireless, remote-controllable soft actuator was also fabricated in a manner similar to that described above, except that no electric wires were connected to the spring.
  • SMA springs with a wire size of 500 pm (Nitinol MicroSprings, Kellogs Research Labs) were used.
  • the actuator was connected to an electric power supply (1403, Global specialties), a circuit board and a microprocessor (Metro 328, Adafruit) that generates electric signals to control the frequency and duty cycle of actuation.
  • the electric circuit is depicted in Figure 7c.
  • the microprocessor transmits the programmed electric signal to the electric power supply through the circuit board, and accordingly the power supply applies a voltage to the actuator to trigger its actuation.
  • a laser of 854 nm wavelength (LD852-SE6OO, Thorlabs) equipped with adjustable collimator lens (LTN33O-A, Thorlabs) was used.
  • a current of 600 mA was applied to the laser to produce the maximum intensity.
  • the laser was focused at a distance of 5 cm.
  • the laser intensity with distance was calculated as the total laser intensity divided by the area of laser spot.
  • the total laser intensity was measured using an optical power meter (PM 160, Thorlabs), and the laser spot area was visualized using a VIS/IR detector card (VRC2, Thorlabs).
  • the soft actuators with varying stiffness were connected to an electric power supply (1403, Global specialties) to apply voltage.
  • the actuation of soft actuators was video-recorded and post-processed using Image J and Tracker.
  • the actuation strain was calculated as the ratio of the change in the length of SMA spring to the initial length.
  • Thermal measurements were performed using a thermal imaging camera (C2, FUR). The camera was placed and focused ⁇ 15 cm away from the actuator. The thermal data was recorded and analyzed with the software (FLIR Tools plus). Lines were drawn either along the axis across the center or along the edge of actuator, and then temperature was measured along those lines. To investigate thermal responses to different frequencies, cyclic actuation was performed with varying frequencies, and the temperature along the edge of actuator was measured and reported as an averaged value. To examine the effect of the surrounding temperature, a chamber filled with distilled water was used and heated to 37 °C to provide a physiologically relevant temperature. It should be noted that the actuator was half immersed in the water, since near-infrared rays cannot penetrate water. Thermal responses to different actuation frequencies were recorded, and the temperature along the edge of actuator was averaged. Thermal imaging of the remotely controlled actuator was also conducted similarly to that described above.
  • hydrogels For hydrogels, cyclic tests were performed to determine their mechanical properties under multiple compressive loads. Hydrogels were first immersed in DMEM containing physiological calcium levels (2.5 mM) for 24 hours and then subjected to 500 cyclic tests with a deformation rate of 0.9 mmsec 1 . The elastic modulus for each cycle was measured as the slope of the stress-strain curve at a strain of 5%. It should be noted that the hydrogels were surrounded by the DMEM containing 2.5 mM calcium during the testing to prevent their dehydration.
  • strains were imposed on the spring ends (i.e., the squares). Given that the actuators were operated at the same voltage in the experiment, the total strain energy in the simulation was first kept the same for all stiffnesses. The strain energy was determined as a value that closely matched the simulated strains and the experimental strains. With this strain energy, the simulation strains generally matched the experimental data, except for the lowest actuator stiffness. This soft elastomer could not maintain a tight bond with SMA due to the low polymer density, and was observed to slip during actuation in the experiment. Therefore, for this stiffness, the strain energy was adjusted to match the simulation strain and the experimental strain. The resulting strain energies were used in subsequent simulations to predict tissue deformation.
  • a simulation model combining the actuator model described above, a tough adhesive model with length of 80 [in arbitrary units (A.U.)] and varied height, a tissue model with length and height of 160 and 40 [in arbitrary units (A.U. )] was constructed with assumption of no slip and friction between the interfaces. Strains that generated the strain energies obtained above were imposed, and tissue deformation was evaluated based on the maximum displacement divided by the initial length of the elastomer. The effect of elastomer stiffness, adhesive stiffness, adhesive thickness and tissue stiffness on tissue deformation was tested.
  • Hydrogel tissue phantoms were prepared based on tough hydrogels with the previously established formulation 26 . Briefly, sodium alginate and acrylamide were dissolved in HBSS at 2% and 12% respectively and stirred overnight until a clean solution was obtained. This solution of 2 mL was then mixed with 7.2 pL of 2% N,N'-Methylenebisacrylamide (MBAA), 1.6 pL of Tetramethylethylenediamine (TEMED), 45.2 pL of 0.27 M Ammonium persulfate (APS), and 38.2 pL of 0.75M CaSCE slurries. The mixture was gelled in a closed mold at room temperature overnight.
  • MBAA N,N'-Methylenebisacrylamide
  • TEMED Tetramethylethylenediamine
  • APS Ammonium persulfate
  • CaSCE 0.75M CaSCE
  • Tough hydrogels were bonded to the elastomer component of the actuator following a previously established method 29 . Briefly, the surface of the elastomer was treated by absorbing benzophenone. Prior to treatment, the surface was thoroughly cleaned with methanol and deionized water, and dried. Benzophenone solution (10 wt% in ethanol) was then applied onto the actuator by covering the entire surface for 5 minutes at room temperature. The actuator was washed 2-3 times with methanol and completely dried.
  • Tough hydrogels were prepared by mixing an aqueous pre-gel solution (12% acrylamide, 2% sodium alginate (LF20/40 and 5 Mrad at 1:1 ratio), 0.037% MBAA, 0.2% Irgacure 2959) with ionic crosslinker (35 mM calcium sulfate).
  • ionic crosslinker 35 mM calcium sulfate.
  • MVG and VLVG, Pronova ultrapure alginates
  • hydrogels were then gently removed from the mold and assembled with benzophenone-treated elastomer followed by UV irradiation (UVP Blak-Ray, 100W) for 20- 30 minutes to crosslink the polyacrylamide network and to form bonds on the elastomer surface.
  • UV irradiation UV Blak-Ray, 100W
  • the adhesion performance was quantified as the adhesion energy, the amount of interfacial energy required to propagate a unit area of interfacial crack.
  • the adhesion energy was measured by T-peeling tests. Samples of the elastomer-hydrogel or hydrogel-tissue interface were prepared by placing and attaching a hydrogel between two elastomers or a tissue between two hydrogels.
  • the back side of the elastomers or hydrogels were bonded to a rigid, non-stretching film (e.g., polyethylene terephthalate (PET)) with Krazy glue to prevent its stretching along the peeling direction, ensuring that all the work done by the mechanical tester was used for the energy dissipated at the crack tip.
  • PET polyethylene terephthalate
  • the free ends of the elastomers or hydrogels were fixed to the grips of the machine. Unidirectional tension was applied with an Instron (3342, load cell of 50N) while recording the force and the extension. The loading rate was kept constant at 50 mm/min.
  • the adhesion energy was determined as twice the plateau force divided by the width of the sample.
  • MAGENTA application was performed based on the previously described method 26 by adding a bridging polymer (z.e., chitosan) onto the tough hydrogel of actuatorhydrogel hybrid and then adhering it to the muscle tissue.
  • a bridging polymer z.e., chitosan
  • 4% ultrapure chitosan (54046, Heppe Medical Chitosan) and coupling reagents (24 mg/mL of l-ethyl-3-(3- dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide) were mixed at 1:1 ratio by vortexing. The mixture was then quickly applied to the surface of the tough hydrogel, and the applied MAGENTA was gently compressed for 5 minutes to achieve tissue adhesion.
  • cytotoxicity tests C2C12 cells were cultured in the growth medium, DMEM containing 10% FBS and 1% Pen strep. When the confluency of cells reached 60 - 70%, cells were cultured in the presence of the soft actuator with tough gel or MAGENTA. Pristine culture medium was used as a control. After one day, cytotoxicity was determined using a live/dead viability kit (Thermo Fisher Scientific) by adding 4 pM calcein and ethidium homodimer- 1 into the medium. Fluorescence images were taken through GFP and RFP channels in EVOS-FL (AMG).
  • MAGENTA In vivo biocompatibility was tested via implantation on muscle. MAGENTA was applied to bicep femoris muscles. After 2 weeks of implantation, MAGENTA was gently removed from muscles, and the muscles were collected and analyzed through histology. In addition, the impact of stimulation on biocompatibility was also assessed. MAGENTA was applied to the bicep femoris muscles of healthy mice, and mechanical stimulation was performed for one week. The muscles were collected and analyzed by histology and immunofluorescence.
  • Gelatin and collagen-based hydrogels were prepared by mixing a pre-gel solution of 19.85% acrylamide, 4.96% gelatin, 1.34 mg/ml collagen, and 0.0149% MBAA with the initiator and accelerator (0.26M APS and 2.44 v/v% TEMED, respectively), and used as culture substrates that can tolerate repeated mechanical deformation by MAGENTA.
  • M AGENTA was applied to the hydrogels, and C2C12 cells were seeded on the remaining area of the gels.
  • For the proliferation assay cells were allowed to spread for one day, and then stimulated at the frequency of 0.1 Hz with maximum strain for 30 minutes per day for 2 days.
  • the fraction of proliferating cells was determined as the percentage of Ki67 positive cells.
  • cells were first cultured on the gels in the regular medium without stimulation until the confluency reached 80 - 90%, and then the medium was changed to DMEM containing 2% horse serum and 1% Pen strep to induce differentiation. Stimulation was performed in the same manner as the proliferation assay for 2 days.
  • the fusion index was defined as the percentage of total cellular nuclei located inside myotubes, as indicated by MHC staining, in relation to the total number of nuclei.
  • hindlimb immobilization was performed in female C57BL6/J mice at 14-18 weeks of age (Jackson Laboratory) following the previously established method with some modifications 75 .
  • the hindlimb was first shaved prior to immobilization, and on the day of immobilization, animals were anesthetized using isoflurane inhalation.
  • One hindlimb was maintained in a knee joint extension and ankle plantar flexion position, and fixed by wrapping with the non-elastic surgical bandages (Micropore surgical tape and Transpore surgical tape, 3M) to the foot.
  • the forefoot portion was left exposed to monitor adverse events. This surgical taping was maintained for either 2 weeks or 3 weeks depending on the experimental setting. Since hindlimb immobilization was performed on only one hindlimb, the other limb allowed the animal to remain ambulatory. All animal procedures were performed in accordance with the Harvard University Faculty of Arts and Sciences Institutional Animal Care and Use Committee guidelines.
  • mice were anesthetized with isoflurane during mechanical stimulation, and untreated mice were also placed in the isoflurane chamber to keep the amount of exposure to the anesthetic agent the same for the groups.
  • the surgical tapes were gently removed from the hindlimb, and an electric power supply (1403, Global specialties) was connected to the MAGENTA.
  • the voltage that produced the maximum actuation strain was applied ( ⁇ 6 V).
  • Actuation frequency was selected to the maximum value at which the temperature of MAGENTA does not exceed the physiological range (0.1 Hz, and duty cycle of 4% on and 96% off).
  • MAGENTA-impl anted animals were anesthetized, placed on a heating pad, and the hindlimbs were fixed to prevent movement. Muscles were analyzed with photoacoustic imaging using a Vevo 770 scanner with a 35 MHz transducer (VisualSonics, axial resolution: 50pm, lateral resolution, 140pm). The scanner was placed directly above where the MAGENTA was located. During ultrasound imaging in the sagittal plane, voltage was applied to the MAGENTA to enable its actuation. After imaging, animals were subsequently allowed to recover to their normal behavior. Images captured were analyzed to estimate muscle displacement driven by the actuation of MAGENTA. Kymographs were created using ImageJ with a stack of ultrasound images at different time points. A line was drawn across a noticeable tissue signal along the MAGENTA axis. Image pixels that the line passed through were then reorganized to create a kymograph.
  • mice were atrophied for 2 weeks via hindlimb immobilization, as described above. Throughout the immobilization, the tibialis anterior (TA) muscle of mice were treated daily with compressive stimulation using a previously developed robotic system 19 (0.3 N, frequency of 1 Hz, duty cycle of 80% for 5 min per day). The photographic images of the system are presented in Figure 26.
  • the robotic device consists of an electromagnetic actuator (DDLM-019-044-01, Moticont) to provide the actuation force, and a force sensor (Honeywell) covered with soft interface to allow continuous measurement of the applied compressive load for feedback control.
  • the force signal measured by the sensor was acquired by an analog input module (NI 9205, National Instruments) and processed by an instrumentation amplifier (UV-10, Honeywell).
  • a real-time controller (cRIO-9030, National Instruments) with a proportional-integral control algorithm was used to determine the force output based on the desired input force.
  • the digital output signal was converted to an analog output using a digital-to-analog module (NI9264, National Instruments), and then transmitted to a motor driver (950 series, MotiCont) to control the actuator.
  • the device was calibrated using a precision scale (Ohaus Scout).
  • the muscle function of isolated lateral gastrocnemius and tibialis anterior muscles was measured as previously described 16,19 . Briefly, explanted muscles were placed between two steel electrodes (1.6 mm diameter, 21 mm long) and fixed with micro-clippers at both ends. The upper of the two clippers was connected to a force transducer (FORT 25, WPII). During muscle function tests, the muscles were bathed in a physiologic saline solution chamber continuously supplied with oxygen at 37°C. Electric stimulation of the muscles and data acquisition were performed through LabVIEW. A wave pulse was initiated and delivered to the electrodes via power amplifier (QSC USA 1310). Tetanic contraction was evoked at 30, 35, and 40V with frequencies of 10 Hz/V.
  • a constant pulse width of 2 milliseconds and a train duration of 1 second were used.
  • the muscles were rested for 5 minutes between the stimulations.
  • Maximum tetanic force was defined as the difference between the maximum force during stimulation and the baseline.
  • Each muscle weight was used for normalization.
  • the sectioned samples were stained using standard immunohistochemistry protocols.
  • the samples were permeabilized with PBS containing 0.1% Tween 20 or Triton X (PBST), blocked with PBST containing 10% bovine serum albumin and 10% goat serum.
  • PBST Triton X
  • laminin antibody (abl l575, Abeam), MRTFA (PA5- 109960, Thermo Fisher Scientific), YAP (ab205270, Abeam), MyoD (ab212662, Abeam), Pax7 (abl87339, Abeam), Ki67 (abl5580, Abeam), MHC (MAB4470, R&D systems), Desmin (abl5200, Abeam), CD31 (abl24432, Abeam), Prolong Gold antifade reagent (Invitrogen) and D API (Invitrogen).
  • tile scanning (Zeiss, LSM710) was performed to capture the entire longitudinal section of the muscle.
  • Regions of interest (ROIs) of 500 pm x 500 pm square were randomly selected throughout the section, and the fractions of cells positive for YAP and MRTFA were measured.
  • Tissue depth was defined as the horizontal distance between the center of the ROI and the edge of the muscle section on the stimulation side.
  • Isolated skeletal muscles were lysed in tissue protein extraction buffer (78510, Thermo Fisher Scientific) supplemented with phosphatase inhibitors cocktail (1861281, Thermo Fisher Scientific) and 0.1 mg/ml of phenylmethylsulphonyl fluoride. Protein concentrations were quantified using Pierce Bicinchoninic Acid Protein Assay Kit (23227, Thermo Fisher Scientific) as per the manufacturer’s instructions. Laemmli sample buffer (1610747, Bio-Rad) was added to lysates, and samples were boiled for 10 minutes before loading in a polyacrylamide gradient gel (4561086, Bio-Rad).
  • Proteins were separated and transferred to nitrocellulose at 100V for 1 hour, blocked with 4% non-fat milk in TBS-T, incubated overnight with primary antibodies followed by incubation for 1 hour at RT with horseradish peroxidase-conjugated secondary antibodies and detection using enhanced chemiluminescent detection reagent (34577, Thermo Fisher Scientific).
  • the following primary antibodies were used for immunoblotting; YAP antibody (ab205270, Abeam), MRTFA antibody (PA5- 109960, Thermo Fisher Scientific), MuRFl (sc-398608, Santa Cruz), Akt (4685S, Cell Signaling Technology), phospho-Akt (4060T, Cell Signaling Technology), GAPDH (ab9485, Abeam), and puromycin antibody (12D10, Millipore,).
  • Secondary antibodies used were goat anti-mouse IgG2a conformation specific antibody (115-035-206, Jackson Immuno Research) for puromycin, goat anti-mouse for MuRFl (405306, BioLegend), and donkey anti-rabbit (406401, BioLegend) for the rest of proteins. Densitometry was performed using ImageJ, and the protein levels were normalized to those of GAPDH. Uncropped scans of western blots are provided in the Figure 30.
  • Protein synthesis was analyzed using SuNSET 58 . Briefly, the mice were injected with 0.04 pmol/g 1 body weight of puromycin (P8833, Sigma) dissolved in PBS through an intraperitoneal injection. At 30 minutes after injection, muscles were collected. The tissues were processed as described above for protein extraction and immunoblot. To control for variation in sample loading, samples were loaded twice; one lane for puromycin and the other lane for GADPH normalization.
  • Muscle tissues were first processed as described above for protein extraction.
  • the protein concentration of IGF-1 in the extraction was quantified using the IGF-1 Mouse ELISA Kit (EMIGF1, Thermo Fischer Scientific) along with recombinant mouse IGF-1 as a standard, and according to the manufacturer’s instruction.
  • Hyperspectral Imaging was carried out with a Horiba XploRa system equipped with the Cytoviva Darkfield in a reflected light mode.
  • the system consists of a Dolan Jenner lamp with a 150W halogen bulb (L1090, International Light Technologies) with an aluminum reflector for enhanced near infrared light delivery.
  • L1090 150W halogen bulb
  • an Olympus BX41 upright microscope was used with x50 objective.
  • the light was sent to a spectrometer (V10E, Specim) through a liquid light guide (77634, MKS-Newport).
  • the reflected light was collected with a CCD camera (PCO Pixelfly), and the light intensity from each pixel was averaged to estimate the reflectance of SMA spring.
  • the reflectance of SMA spring was normalized relative to that of a silver mirror.
  • the elastic modulus of elastomers was estimated using the neo-Hookean model with incompressibility.
  • the neo-Hookean model with incompressibility can be expressed as where U is the strain energy density, the principle stretch, p the shear modulus.
  • the engineering stress of the incompressible neo-Hookean model with uniaxial deformation can be described as
  • the shear modulus of elastomer was then determined by fitting the equation with the experimental data. As shown in the Figure 8a, the neo-Hookean model well captured the experimentally measured elastomer stress. Finally, the elastic modulus of elastomers was determined as three times the shear modulus.
  • E eiec , E strain , E thermai and Ef riction represent the electrical, strain, thermal and friction energy, respectively.
  • E eiec , E strain , E thermai and Ef riction represent the electrical, strain, thermal and friction energy, respectively.
  • Ef riction should be negligible, Efriction ⁇ 0 ⁇
  • E strain is identical for all elastomer stiffness.
  • the calculated strain energy closely matched the experimentally measured strains ( ⁇ 7000 J/m°, black open circles in the Figure 10b) over most values of the elastomer stiffness.
  • the calculated strain energy did not match the experimental strain at the lowest elastomer stiffness. This soft elastomer could not maintain a tight bond with SMA probably due to the low polymer density and was observed to slip during actuation in the experiment. Therefore, the assumption of no slip was not valid for this stiffness, and Ef riction is no longer negligible.
  • the strain energy was adjusted such that the simulation strain can capture the experimental strain ( ⁇ 3000 J/m 3 , in the Figure 10b). With this adjusted strain energy, the simulation strain finally matched the experimental strain.
  • strain energy in this simulation is the sum of the strain energy of each component. where and represent the strain energy of actuator, adhesive, and tissue, respectively. Since the electrical, thermal, and friction energies are the same as with the actuator alone, the strain energy, of this simulation should also be the same as the strain energy determined above. Simulations were then performed while maintaining constant strain energy with varying elastomer stiffness, adhesive stiffness, adhesive thickness and tissue stiffness (except for the lowest elastomer stiffness as explained above).
  • the actuation strain defined as the ratio of the change in spring length to the initial length, was controlled in a voltage-dependent manner until it reached the maximum value ( Figure 1c and d).
  • the contracted actuator relaxed promptly to its initial state, driven by the restoring force of the elastomer, allowing for consecutive actuation ( Figure 1c).
  • the frequency and duration of actuation can be simply programmed through a microprocessor ( Figure 7c). Given the simplicity of the fabrication process, the design and size of the actuators can be readily scaled from millimeters to several centimeters ( Figure 7d). This approach established a simple yet facile method to build soft robotic actuators that can provide programmed actuation on demand.
  • the actuator-adhesive hybrid was prepared by chemically anchoring the elastomer component of the actuator to the hydrogel adhesive (Figure 3a). As the adhesive matrix will undergo cyclic deformation during actuation, cyclic compression tests were performed to determine its impact. The stiffness of the hydrogels declined during the first few compressions and became stabilized over the next hundreds of compressions ( Figure 3b). We defined the stiffness of the hydrogels as the average value over 500 cyclic tests. On the basis of the simulation results, the chemical and physical crosslinking density of the gels was tuned such that the stiffness value was in the range of 30 - 60 kPa (Figure 3c), which was predicted to generate the maximum tissue strain (Figure 2j).
  • the hydrogel was then chemically bonded to the elastomer of the actuator using benzophenone 29 .
  • the other face of the hydrogel was anchored to tissue with a chitosan bridging polymer (see the Materials and Methods).
  • the actuator-hydrogel hybrid with tissue adhesion is hereafter referred to as the mechanically active gel-elastomer-nitinol tissue adhesive (MAGENTA).
  • MAGENTA mechanically active gel-elastomer-nitinol tissue adhesive
  • MAGENTA To demonstrate the ability of MAGENTA to deliver therapeutic mechanical stimulation, MAGENTA was first applied to a phantom hydrogel tissue (Figure 3g; Figure l la-c). Upon applying voltage, the MAGENTA contracted, and the underlying phantom tissue was deformed accordingly ( Figure l la-c). MAGENTA was next applied to skeletal muscle tissue ex vivo, and the MAGENTA contraction again led to the deformation of the underlying tissue ( Figure 3h; Figure l id). The experimentally measured tissue strain, determined by tracking the paint speckles on the surface of excised muscle tissues, was similar to that predicted by the simulation ( Figure 2h-k and Figure 3i; Figure lOe and f).
  • MAGENTA The biological compatibility of MAGENTA was initially analyzed by coculturing a MAGENTA construct with C2C12 myoblasts. No significant decrease in cell viability was found with MAGENTA exposure ( Figure 15a and b). MAGENTA was subsequently implanted into mice, and histological assessment demonstrated that it generated minimal to mild inflammation ( Figure 15c and d). Next, the impact of stimulation on biocompatibility was assessed. MAGNETA was applied to muscles of healthy mice, and mechanical stimulation was performed. Similar to the muscles treated with MAGENTA alone, stimulated muscles also showed only minimal to mild inflammation, and no significant effects were observed on tissue homeostasis, including cell proliferation and vascularization ( Figure 7).
  • mice treated with stimulation had muscle fibers with larger cross-sectional area and greater levels of MHC and desmin expression, as compared to untreated mice ( Figure 5, a-c; Figure 21, c-h).
  • the fractions of cells positive for Ki67 and Pax7, indicative of proliferating cells and satellite stem cells, were also higher with mechanical stimulation.
  • the gross size and weight of muscles were also measured greater in stimulated mice ( Figure 5d and e). However, no significant increase was observed in matrix protein deposition with stimulation (Figure 22).
  • mice had similar body weight after 2 weeks (Figure 5f). Muscle function analysis demonstrated that the muscles of treated mice generated substantially higher tetanic forces than those of untreated mice, with force levels similar to those of muscles in healthy, active mice ( Figure 5g and h).
  • mice were first atrophied for 3 weeks (Figure 25a), and then mechanical stimulation was performed for the subsequent week. Similar to the prevention experiments, mechanically stimulated muscles displayed greater weight and tetanic force generation compared to those untreated ( Figure 25, b-f).
  • the light responsive actuator was then used to create a wireless, remote-controllable MAGENTA (Figure 27f).
  • Ex vivo experiments demonstrated, similar to the electric MAGENTA, that actuation of wireless MAGENTA substantially deformed the underlying muscle tissue along the actuator axis ( Figure 6e and f).
  • the wireless MAGENTA was observed to contract following laser irradiation through the mouse skin ( Figure 6g), demonstrating the remote delivery of mechanical stimulation.
  • the responsiveness of MAGENTA versus implantation depth was evaluated. The wireless MAGENTA was first adhered to excised muscle tissues, and porcine skin and muscle tissues were then placed on top of the MAGENTA to simulate varying implantation depth ( Figure 28).
  • MAGENTA a new class of biomedical device capable of firmly adhering to a target tissue and delivering muscle- contraction-mimicking mechanical stimuli in a highly controlled manner to promote tissue regeneration and rehabilitation.
  • the therapeutic effects of this mechanical stimulation were demonstrated in a disuse muscle atrophy model. With mechanical stimulation, the occurrence of atrophy was delayed, and recovery was greatly facilitated.
  • Efficient mechanical stimulation of tissues requires both a mechanism of actuation and robust adhesion to efficiently deliver the mechanical cue to the target tissue.
  • This study used SMA springs to provide controlled actuation, and tough adhesives to achieve strong tissue adhesion. While SMA springs actuate at elevated temperatures, the elastomer surrounding the spring acts as an insulator to protect the tissue. Guided by computational simulations, we determined the optimal combination of actuator stiffness (z.e., -130 kPa), adhesive stiffness (i.e., 30 - 60 kPa) and thickness (the thinner the better) to produce maximum tissue deformation.
  • the resulting deformation in skeletal muscle was measured at 15 - 20%, which has been shown to generate substantial activation of muscle cells in previous in vitro studies 43,44 , and also did so in our in vitro model.
  • the biocompatibility of MAGENTA was confirmed in vitro and in vivo, and is consistent with previous studies employing tough adhesives 26,45 .
  • MAGENTA In addition to mechanical stimulation, MAGENTA generates moderate heating upon actuation, which possibly provides additional benefits to the muscles, as suggested in previous studies of thermo therapy 64 ” 66 .
  • thermo therapy 64 thermo therapy 64
  • MAGENTA As a potentially important function of MAGENTA, we demonstrated wireless, remote-controlled actuation. While remote control systems typically involve complex electrical circuits, such as near field communication (NFC) 69 71 and radio frequency (RF) system'' 2,73 , and require a large physical space to mount these additional equipment, wireless actuation of MAGENTA is possible without additional electric components or complex application systems. This was enabled by the light absorption of SMA and consequent thermal generation. The laser- driven actuation of SMA has not been previously studied. However, compared to the electric MAGENTA, wireless MAGENTA has a limited actuation frequency due to the time of thermal generation required with laser irradiation.
  • NFC near field communication
  • RF radio frequency
  • MAGENTA provides an effective approach to mechanical stimulation for tissue regeneration and rehabilitation.
  • One benefit of MAGENTA is its simple fabrication methods and low-cost materials, potentially allowing for low-cost mass production. While the current work was mainly focused on skeletal muscle stimulation in the context of disuse atrophy, this system may also be directly applicable to other types of tissues, including skin and heart ( Figure 29).
  • the ability to remotely control MAGENT A may also further extend its use in a wide array of applications, ranging from soft robotics to regenerative medicine.
  • the IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy- induced ubiquitin ligases by inhibiting FOXO transcription factors. Mol. Cell 14, 395- 403 (2004). Jacques, S. L. Optical properties of biological tissues: A review. Phys. Med. Biol. 58, R37-R61 (2013). Nakai, N., Kawano, F. & Nakata, K. Mechanical stretch activates mammalian target of rapamycin and AMP-activated protein kinase pathways in skeletal muscle cells. Mol. Cell. Biochem. 406, 285-292 (2015). Lin, S. S. & Liu, Y. W.

Abstract

La divulgation concerne un adhésif de tissu de type gel-élastomère-nitinol mécaniquement actif (MAGENTA) qui peut générer et administrer une stimulation imitant une contraction musculaire à un tissu cible avec une intensité et une fréquence programmées. Le MAGENTA comprend un actionneur souple, qui comprend un alliage à mémoire de forme et un élastomère, et un adhésif qui fait adhérer l'actionneur au tissu sous-jacent. Il a été déterminé que le MAGENTA active des voies de mécanodétection impliquant la protéine associée à Yes (YAP) et le facteur A de transcription associé à la myocardine (MRTFA) lorsqu'il est fixé au muscle, et augmente le taux de synthèse de protéines. Les muscles inactifs traités avec le MAGENTA ont présenté une taille et un poids supérieurs et, de manière importante, ont permis de générer des forces significativement supérieures par rapport aux muscles non traités, ce qui démontre la prévention de l'atrophie. Enfin, l'actionnement du MAGENTA peut être commandé à distance, ce qui élargit la portée de ses applications potentielles.
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