Compliant mechanism for enhancing and / or decoding performance of tissues

Abstract

This disclosure provides a device for assessing forces in tissues and methods of use thereof. The device may be used, for instance, to probe forces associated with characteristics to regulate spatial displacement of a tissue in one or more directions. The device may have at least two rigid attachment points for supporting a tissue and connected to a compliant mechanism to allow displacement of a tissue in a direction x, wherein tissue displacement is constrained in at least one direction other than x. The device may have a compliant mechanism with a stiffness value in a displacement direction that is lower than a stiffness value of the tissue in the displacement direction and / or at least one of the rigid attachment points having a stiffness value in the displacement direction that is higher than a stiffness value of the tissue in the displacement direction.

Description

[0001] COMPLIANT MECHANISM FOR ENHANCING AND / OR DECODING PERFORMANCE OF TISSUES

[0002] RELATED APPLICATION

[0003] This application claims the benefit of the filing date under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 571,325, filed on March 28, 2024, the entire contents of which is incorporated herein by reference in its entirety.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with government support under W91 INF-22-1-0126 awarded by the U.S. Army Research Office and CMMI- 2238715 awarded by the National Science Foundation. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Current efforts to characterize the performance of engineered tissues rely on flexible pillar-based methods to track contractile displacement and calculate force. However, its accuracy is inherently limited by uncertainties in measuring the placement of muscle along the length of the beam, resultant displacements and motions of the pillars, as well as the contact area between the muscle and pillar. The assumptions made by beam-based calculations of force thus significantly reduce the ability to accurately calculate forces, particularly in response to high- frequency stimulation.

[0008] SUMMARY

[0009] Research methods on tissue encompass a range of techniques aimed at studying its structure, function, composition, and behavior. In particular, functional studies of tissue, such as muscle tissue provide valuable insight into a number of properties of the tissue. For instance, functional studies of tissues, such as muscle, enable assessment of properties such as muscle contraction, force generation, fatigue, and other physiological parameters. The ability to analyze these properties holds great potential for drug screening to identify drugs capable of modulating these properties. For instance, researchers can systematically investigate how drugs interact with muscle tissue and elucidate their potential therapeutic benefits, side effects, and mechanisms of action, ultimately informing clinical practice and drug development efforts.

[0010] Additionally, nearly all machines require components that generate force and produce motion, motivating the development of a diverse array of robust and efficient actuators for a range of engineering applications. Biological actuators such as skeletal, cardiac, and smooth muscle are designed for robust and efficient force generation that senses and responds to dynamic surroundings in real-time.

[0011] Previous efforts to characterize the performance of 3D tissue such as cardiac and skeletal muscle have almost exclusively relied on flexible pillar-based method to track contractile displacement and calculate force. However, the assumptions made by these methods significantly reduce the ability to accurately calculate forces, predict clinical outcomes through research measurements, and prevent the reproducible design, manufacturing, and deployment of living muscles as actuators. In aspects of the disclosure, new tools for accurate, precise, and quantitative performance monitoring of living muscle tissue are disclosed, as well as methods to match and optimize their outputs to the needs of the specific application.

[0012] In some aspects, the techniques described herein relate to a device arranged to produce stiffness characteristics that regulate spatial displacement of a tissue in one or more directions, comprising a solid support comprised of at least two rigid attachment points for supporting a tissue, wherein a first rigid attachment point positioned at one end of the solid support is connected to a compliant mechanism to allow displacement of a tissue and relative to a second rigid attachment point and in a direction x, wherein tissue displacement is constrained in at least one direction other than x, and optionally wherein the compliant mechanism has a stiffness value in the one or more directions that is lower than a stiffness value of the tissue in the one or more directions and at least one of the rigid attachment points has a stiffness value in the one or more directions that is higher than a stiffness value of the tissue in the one or more directions. In some aspects a device, comprising (i) a first attachment point; (ii) a second attachment point adjacent to the first attachment point, such that a tissue can interact with the first attachment point and the second attachment point; and (iii) a compliant mechanism arranged to produce stiffness characteristics having a first end and a second end; wherein the first end of the compliant mechanism is connected to a solid support and the second end of the compliant mechanism is connected to the second attachment point; and wherein the compliant mechanism has a lower degree of stiffness in one direction than at least one other direction is provided.

[0013] In some embodiments, the solid support is a base and wherein the base is attached to the first attachment point and the compliant mechanism, wherein the first attachment point is attached to the base by an adjustment mechanism, optionally wherein the adjustment mechanism is: a) in a fixed position, b) an adjustable cam, a wedge, a screw, or a lever, c) wherein the adjustment mechanism enables the first attachment point to be adjusted in at least one direction or orientation relative to the second attachment point, or d) enables the distance between the first attachment point and the second attachment point to be adjusted to match the passive length of the tissue.

[0014] In some embodiments, the first attachment point is a rigid pillar and / or wherein the second attachment point is a rigid pillar.

[0015] In some embodiments, the compliant mechanism has a rectilinear topology, wherein the rectilinear topology comprises parallel beams conjugated in series, wherein the rectilinear topology yields: a) a low stiffness in one or more directions b) a high stiffness in one or more directions, or c) a low stiffness in one direction and a high stiffness in two directions.

[0016] In some embodiments, the compliant mechanism is made from a linear elastic material and optionally wherein the linear elastic material is a metal, a ceramic, or a biocompatible polymer.

[0017] In some embodiments, the compliant mechanism has a stiffness value in the one or more directions a) that is less than the stiffness value of the tissue in the one or more directions, b) that equals the stiffness value of the tissue in the one or more directions, c) that exceeds the stiffness value of the tissue in the one or more directions, or d) that is higher than a stiffness value of the tissue in the one or more directions. In some embodiments, the linear clastic material is a low- density polyethylene.

[0018] In some embodiments, the first end of the compliant mechanism is connected to the base by a mount.

[0019] In some embodiments, the device further comprises 2-4 additional attachment points capable of interacting with the tissue.

[0020] In some embodiments, the compliant mechanism has a lower degree of stiffness in one direction than in all other directions or wherein the tissue displacement is constrained all directions other than x.

[0021] In some embodiments, the device is monolithic or modular.

[0022] In some embodiments, the device is configured to measure displacement, speed and / or acceleration.

[0023] In some embodiments, the device further comprises an actuator, wherein the actuator is configured to regulate spatial displacement of one or more components of the device.

[0024] In some embodiments, the stiffness of the compliant mechanism in the one or more directions relative to the stiffness of the tissue in the one or more directions is between 0.01 and 100.

[0025] In some embodiments, the first and second attachment points are sized to fit within a well of a multi-well plate, such as a 24 or 96 well plate.

[0026] In some embodiments, the device is sized to function with a single well of a multi-well plate, such as a 24 or 96- well plate.

[0027] In some aspects a method of assessing a tissue with a device is provided. The method comprises (i) contacting a first attachment point and a second attachment point of a device with a tissue; (ii) stimulating the tissue or device to produce a force one or more times; and, (iii) measuring a property associated with displacement of the second attachment point relative to the first attachment point each of the one or more times, wherein the first attachment point and the second attachment point are rigid attachment points; wherein the second attachment point is connected to a base of the device by a compliant mechanism; and wherein the first attachment point is connected to the base of the device and wherein at least one of the rigid attachment points has a stiffness value in the direction of displacement that is greater than or equal to a stiffness value of the tissue in the direction of displacement. In some embodiments, the device is a device as disclosed herein.

[0028] In some embodiments, the first attachment point and the second attachment point are spaced at a fixed distance and wherein the fixed distance matches the passive length of the tissue or wherein the fixed distance is greater than the passive length of the tissue.

[0029] In some embodiments, the tissue is a contractile tissue, optionally wherein the contractile tissue is a skeletal muscle tissue, cardiac muscle tissue, or smooth muscle tissue.

[0030] In some embodiments, the tissue sample is a non-contractile tissue, optionally wherein the tissue is a connective tissue, an epithelial tissue, or a tissue of the nervous system, optionally wherein the connective tissue is a tendon, a ligament, a fibroblast, or cartilage, wherein the epithelial tissue is skin or blood vessels, and wherein the tissue of the nervous system is a motor neuron, glia, or ganglion.

[0031] In some embodiments, the force is a normal force or a shear force.

[0032] In some embodiments, the tissue or device is stimulated optically, such as stimulation with fiber optics, chemically, electrically, mechanically, thermally, or magnetically.

[0033] In some embodiments, the force is produced by the compliant mechanism or by the tissue.

[0034] In some embodiments, displacement can be measured using image tracking.

[0035] In some embodiments, the force can be produced by low-frequency stimulation optionally, wherein the low-frequency stimulation is less than or equal to 1 Hz.

[0036] In some embodiments, the force can be produced by high-frequency stimulation optionally wherein the high-frequency stimulation is greater than or equal to 2 Hz.

[0037] In some embodiments, the property associated with displacement of the second attachment point relative to the first attachment point is measured as displacement, speed, velocity, acceleration, or combinations thereof. A method for evaluating effect of a biologically active substance on a tissue is provided in other aspects. The method comprises (i) contacting a tissue with a biologically active substance; wherein the tissue is positioned on a device and in contact with at least two attachment points on the device, and wherein a compliant mechanism is connected to at least one of the two attachment points on the device, wherein at least one of the first attachment point and the second attachment point have a stiffness in a direction of desired motion that is greater than the stiffness of the tissue in the direction of desired motion; (ii) producing a force one or more times; and (iii) measuring a property associated with displacement of the second attachment point relative to the first attachment point each of the one or more times, wherein the property associated with displacement provides information on the effect of the biologically active substance on the tissue optionally wherein the biologically active substance is a small molecule, a nucleic acid, a gene therapy product, or a protein. In some embodiments, the device is a device as disclosed herein.

[0038] In other aspects a device is provided. The device comprises (i) a solid support comprising a first attachment point and a second attachment point adjacent to the first attachment point, such that a tissue can interact with the first attachment point and the second attachment point; and (ii) a compliant mechanism connected to the second attachment point and structurally between the first and second attachment point so that it takes significant load from that applied by the tissue, and (iii) a sensing mechanism comprised of a strain gauge that is placed at a location within a structural loop between the first attachment point and the second attachment point.

[0039] In some embodiments, the device comprises 1-4 strain gauges.

[0040] In some embodiments, the sensing mechanism is a comprised of a straight beam with four semiconductor strain gauges.

[0041] In some embodiments, the strain gauges are arranged as a bridge circuit, with two of the four semiconductor strain gauges arranged on each end on opposite sides.

[0042] In some embodiments, the device comprises a low-density polyethylene material.

[0043] In some embodiments, the compliant mechanism has a first end and a second end and is arranged to produce stiffness characteristics; wherein the first end of the compliant mechanism is connected to a solid support and the second end of the compliant mechanism is connected to the second attachment point; and wherein the compliant mechanism has a lower degree of stiffness in a direction x that is nominally aligned with the load and displacement of the tissue.

[0044] In some embodiments, the first attachment point allows displacement of the tissue relative to a second attachment point and in a direction x, wherein the relative displacement of the tissue ends is constrained in at least one direction other than x, and optionally wherein the compliant mechanism has a stiffness value in the one or more directions that is lower than a stiffness value of the tissue in the one or more directions, and optionally wherein the relative displacement of the tissue ends is constrained in all other directions other than x.

[0045] In some embodiments, the strain gauge comprises D-type mono-crystalline silicon.

[0046] In some embodiments, the strain gauge comprises metallic strain gauges.

[0047] In some embodiments, the strain gauge has welded gold alloy leads.

[0048] In some embodiments, the structural loop carries a load placed upon it, that is the parts of the structure that feel the force exerted by the muscle.

[0049] In some embodiments, the device includes a tensioning mechanism, wherein the tensioning mechanism is arranged to connect to at least one of the attachment points.

[0050] In some embodiments, the device includes a variable stiffness mechanism, wherein the tensioning mechanism and the variable stiffness mechanism each are arranged to connect to one of the at least two attachment points for supporting a tissue.

[0051] In some aspects a device for regulating spatial displacement of a tissue in one or more directions is provided. The device comprises a base having a circular geometry with an outer perimeter and a grounded central point, a tensioning mechanism and a variable stiffness mechanism, wherein the tensioning mechanism and the variable stiffness mechanism each connect to one of at least two attachment points for supporting a tissue.

[0052] In some embodiments, the device comprises a first attachment point for supporting a tissue, attached at a first position on the outer perimeter of the base and a second attachment point for supporting the tissue, attached at a second position on the outer perimeter of the base and arranged relative to the first attachment point, such that the tissue can interact with the first attachment point and the second attachment point.

[0053] In some embodiments, the tensioning mechanism comprises a tension arm connected to the grounded central point by a rotary tension flexure bearing (TFB).

[0054] In some embodiments, the variable stiffness mechanism comprises a variable stiffness arm and a variable stiffness flexure stage (VSFS), wherein the variable stiffness arm is connected to the grounded central point and also contacts a variable stiffness actuator point (VSAP). In some embodiments, the variable stiffness arm varies in cross-section along its length. In some embodiments, the VSFS and the variable stiffness arm are arranged relative to one another such that a radial gap exists between them. In some embodiments, an actuator interfaces with the VSFS at a variable stiffness actuator point (VSAP).

[0055] In some embodiments, an actuator interfaces with the tensioning arm at a tensioning actuation point (TAP).

[0056] In some embodiments, the device includes an actuator, wherein the actuator has one or two degrees of freedom. In some embodiments, the actuator comprises a motor housing and two shafts, wherein one of the shafts is connected to the actuation point for the TAP and the other of the shafts is connected to the actuation point for the VSAP. In some embodiments, the two shafts are arranged to rotate independent of one another.

[0057] In some aspects a device is provided. The device includes a multi- well plate lid, a first attachment point and a second attachment point adjacent to the first attachment point, such that a tissue can interact with the first attachment point and the second attachment point attached to a bottom surface of the multi-well plate lid or otherwise positioned at the bottom surface of the lid; and; and a compliant mechanism connected to the first attachment point.

[0058] In some aspects, a method of assessing a tissue with a device is provided. The method involves (i) contacting a first attachment point and a second attachment point of a device with a tissue, wherein the second attachment point is connected to a compliant mechanism comprising more than one functional beam which collectively provide a stiffness value; (ii) stimulating the tissue or device to produce a force one or more times; and, (iii) measuring a property associated with displacement of the second attachment point relative to the first attachment point each of the one or more times, wherein the stiffness value of the compliant mechanism is modified and steps (ii) and (iii) are repeated.

[0059] In some embodiments, the stiffness value of the compliant mechanism is reduced by reducing the number of functional beams, physically disrupting the body of the beams or their structural connections.

[0060] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

[0061] BRIEF DESCRIPTION OF DRAWINGS

[0062] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0063] Throughout the disclosure, and in particular in the description of the figures and the examples, muscle is used as an example. Additionally, in some aspects of the description, a flexure is used as an example of a compliant mechanism and a ground pin and a mobile pin are used as examples of the first and second attachment points. However, the invention is not limited to these exemplary embodiments, unless otherwise specified in the claims.

[0064] FIGs. 1A-1E show mathematical model characterization of tissue-device comprising a compliant mechanism interaction. FIG. 1A shows an exemplary schematic for fabricating engineered muscle tissue rings by injection molding a cell / gel suspension of C2C12 myoblasts in a mixture of Matrigel / fibrinogen / thrombin. An exemplary fabricated muscle tissue is shown below the schematic with the inset showing immunohistochemical image of cell nuclei (stained with DAPI, blue) and mature muscle fibers (stained with MYH4, green) distributed uniformly throughout the tissue. FIG. IB shows a schematic of an exemplary muscle ring (2) placed around a first attachment point (4) and a second attachment point (6). In the passive state (State A), the muscle has a length lo and total cross-sectional area Ao. Unconstrained contraction of the muscle to its maximum amount, xp, is represented by State B. State C represents the reduced net movement of the muscle (2), Xi, when an external load is imposed on the second attachment point (6). FIG. 1C shows the compliant mechanism and muscle modeled as springs of stiffness Zyand km, respectively, that exert forces on the second attachment point in response to the muscle actuation force, Fac. FIG. ID shows a free body diagram of the second attachment point (6)demonstrating that muscle actuation is counteracted by the force the muscle expends on compressing itself, Fim, thus reducing the force the muscle exerts on the compliant mechanism to Fme. FIG. IE shows the muscle’s exertion force, is constant, reduced linearly, or follows another decreasing trend, in proportion to the product of tissue stiffness kmand the amount of compressive displacement (red). Compliant mechanism (8) force, Ff, increases linearly as compressive displacement increases (green)).

[0065] FIGs. 2A-2C show fabrication and computational validation of compliant mechanisms (8) with integrated muscle tissue. FIG. 2A is a schematic rendering of an exemplary compliant mechanism (e.g., a flexure (10)), in which precision milling tools are leveraged to fabricate a biocompatible compliant mechanism composed of several thin parallel beams connected to a second attachment point (6), e.g., mobile pin (12). Muscle rings (2) can be wrapped around the first attachment point (4), e.g., ground pin (14) and second attachment point (6), e.g., mobile pin (12), and tissue contraction drives compliant mechanism deformation. FIG. 2B is an image showing a muscle (2) wrapped around pins (12 and 14) of an exemplary compliant mechanism device (e.g., a flexure (10)). Inset shows an optical fiber (22) used to stimulate contraction of optogenetic muscle cells on demand. FIG. 2C shows a finite element analysis model of an exemplary compliant mechanism (e.g., a flexure (10)) showing that exerting an x-direction force, F, on the mobile pin (12) (simulating muscle contraction) yields large deformations in the x axis with relatively low displacement in and about the y and z axes, respectively.

[0066] FIGs. 3A-3F demonstrate that compliant mechanisms (8) disclosed herein can be used to enhance muscle stroke and enable precise reproducible displacement measurement. FIG. 3A shows a schematic of a free muscle (2) in its passive and stimulated state. Arrows show that the direction of muscle contraction is unconstrained throughout the tissue. FIG. 3B shows a schematic of muscle (2) wrapped around a prior art device comprising elastomeric beams (24) in passive and stimulated states. FIG. 3C shows a schematic of muscle wrapped (2) around ground (14) and mobile (12) pins of an exemplary compliant mechanism (e.g., a flexure (10)). Arrows show that muscle contraction direction is constrained by the compliant mechanism design. FIG. 3D shows a comparison of contractile strain across free, beam, and flexure modes of measuring muscle displacement in response to stimulation and demonstrates that compliant mechanisms disclosed herein significantly enhance muscle stroke as compared to the unconstrained and beam-based measurement methods. Unpaired t-test, n=4 per group, * indicates p < 0.05, ** indicates p< 0.005, **** indicates p < 0.0001. FIG. 3E shows contractile displacement of muscle formulated from low growth factor (GF) and high GF Matrigel measured on beams. No significant difference is observed due to large inter-device variability in measurement and low stroke. Unpaired t-test, n=6 per group, ns indicates not significant. FIG. 3F shows contractile displacement of muscle formulated from low GF and high GF Matrigel measured on an exemplary compliant mechanism. Muscle contractile displacement on compliant mechanisms disclosed herein enables large strokes and precise measurement that is insensitive to muscle placement on the device. Unpaired t-test, n=5 per group, **** indicates p < 0.0001.

[0067] FIGs. 4A-4H show contractile dynamics modulation in muscle actuators. FIG. 4A shows stable monitoring of muscle contraction enables measurements of response time following stimulation, contraction time to peak displacement, time at peak displacement, and relaxation time to baseline. FIG. 4B shows average values of response, contraction, peak, and relaxation times for three representative muscle tissues. FIG. 4C shows muscle displacement as a function of time for a tissue stimulated at 1 Hz with 20%, 50%, and 80% stimulus duty cycle and demonstrates that time at peak displacement is modulated by light pulse dynamics. FIG. 4D shows a comparison of time at peak displacement across different duty cycles for 1Hz stimulation, demonstrating that contraction dynamics can be significantly and reproducibly modulated by methods disclosed herein. Unpaired t-test, n=5 per group, * indicates p < 0.05, **** indicates p < 0.0001. FIG. 4E shows muscle displacement as a function of time for a tissue stimulated at 2 Hz with 20%, 50%, and 80% stimulus duty cycle. FIG. 4F shows a comparison of time at peak displacement across different duty cycles for 2 Hz stimulation. Unpaired t-test, *** indicates p < 0.001, **** indicates p < 0.0001. FIG. 4G shows muscle displacement as a function of time for a tissue stimulated at 4 Hz with 20%, 50%, and 80% stimulus duty cycle, demonstrating that time at peak displacement is modulated by light pulse dynamics and that tetanic contraction is induced at 80% duty cycle. FIG. 4H shows a comparison of time at peak displacement across different duty cycles for 4 Hz stimulation, demonstrating that contraction dynamics can be significantly and reproducibly modified by methods disclosed herein. Unpaired t-test, n=5 per group, ** indicates p < 0.01, *** indicates p < 0.001.

[0068] FIGs. 5A-5G show muscle force, work, and power output as a function of stimulus frequency. FIG. 5A shows predicted force displacement relationship for muscles based on measured values of maximum untethered displacement, muscle stiffness, and compliant mechanism stiffness. FIG. 5B shows a comparison of predicted and observed contraction, demonstrating that muscles significantly outperform predicted displacement because compliant mechanisms disclosed herein constrain the tissue’s degree of freedom of motion during contraction. Unpaired t-test, n=5 per group, * indicates p < 0.05. FIG. 5C shows observed contractile forces on an exemplary compliant mechanism are significantly higher than predicted muscle force output based on unconstrained tissue measurements. Unpaired t-test, n=5 per group, * indicates p < 0.05. FIG. 5D shows force output over time in response to 1 Hz stimulation for three representative muscle tissues. FIG. 5E shows force output over time in response to 4 Hz stimulation for three representative muscle tissues. FIG. 5F shows a comparison of work output per cycle for muscle stimulated at 1 Hz and 4 Hz, demonstrating significantly higher work generated per contraction cycle at lower frequency. Unpaired t-test, n=3 per group, ** indicates p < 0.01. FIG. 5G shows a comparison of power output for muscle stimulated at 1 Hz and 4 Hz, demonstrating no significant difference despite greater variability in power output in response to higher frequency stimulation. Unpaired t-test, n-3 per group, ns indicates not significant.

[0069] FIGs. 6A-6B shows frequency-dependent fatigue in muscle. FIG. 6A shows a comparison of muscle contractile stroke after 0, 15, and 30 minutes of continuous 1 Hz 20% duty cycle stimulation. No significant change in displacement is observed. Unpaired t-test, n=3 per group, ns indicates not significant. FIG. 6B shows a comparison of muscle contractile stroke after 0, 15, and 30 minutes of continuous 4 Hz 20% duty cycle stimulation. A significant drop in displacement is observed after 15 and 30 minutes. Unpaired t-test, n=3 per group, * indicates p < 0.05, **** indicates p < 0.0001.

[0070] FIG. 7 is a diagram showing the use of a flexure device in a multi-well plate. The mobile pin (12) and fixed pin (14) are fixed to the lid and when the lid is placed on the multi- well plate (30) the pins are inserted into a well (32), where the tissue can be formed around the pins.

[0071] FIGs. 8A-8D is a series of diagrams depicting an embodiment of the flexure device in which the device has a circular architecture, also referred to as a Variable Stiffness Muscle Meter. FIG. 8A depicts muscle placement (34) on pins (12, 14) on a variable stiffness muscle meter. FIG. 8B depicts an example of rotation of tensioning arm (36) showing displacement of the upper muscle attachment pin (12). FIG. 8C depicts various elements of variable stiffness muscle meter, including a variable stiffness arm (38), a variable stiffness flexure stage (VSFS) (40), a variable stiffness flexure bearing (VSFB) (42), and a variable stiffness actuator point (VSAP) (44). FIG. 8D depicts actuated motion of the VSFS.

[0072] FIG. 9 depicts a candidate actuation scheme for two-shaft actuation of variable stiffness muscle meter.

[0073] FIG. 10 depicts a candidate actuation scheme for variable stiffness muscle meter.

[0074] FIG. 11 depicts the movement of an actuation scheme for variable stiffness muscle meter, where the motor pushes the tensioning arm to a fixed point at A, releases it and then rotates through the arc.

[0075] FIG. 12 depicts a CAD model of a flexure device.

[0076] FIG. 13 is a CAD model of flexure bank.

[0077] DETAILED DESCRIPTION

[0078] Methods for characterizing the performance of contractile tissues have previously been performed using a flexible pillar-based method to track contractile displacement and calculate force. While these methods have enabled some advancement in the field, the accurate measurement and calculation of forces has been challenging. In contrast to the limitations of existing techniques to measure the forces generated by living tissues, devices that: 1) reproducibly monitor tissue motions and forces with fine accuracy and precision without severely limiting muscle contraction; 2) are insensitive to changes in the geometry and placement of muscle tissues that are a natural consequence of using nonhomogeneous biological materials; and, 3) can be manufactured in multiple ways (i.e. monolithic, modular etc.) are provided herein, as well as methods of use thereof.

[0079] Accordingly, aspects of this disclosure provide a device capable of producing and modifying stiffness characteristics to regulate spatial displacement of a tissue in one or more directions. Unlike conventional mechanisms, devices of the present disclosure rely on sub- optimal geometries combined with the inherent properties of their material (e.g., modulus, viscoelasticity) to accomplish their task. Additional aspects of the disclosure include devices capable of providing variable stiffness levels to achieve variability in displacement and analysis as well as devices with integrated sensors to enhance efficiency and quality of measurements. In various aspects the devices are also optimally designed such that they are compatible with and for use with standard multi-well plates. Other aspects of this disclosure provide a method of assessing a tissue with a device provided herein. Additional aspects of this disclosure provide a method for evaluating the effect of a biologically active substance on a tissue.

[0080] Devices

[0081] In some embodiments, this disclosure provides a device arranged to produce stiffness characteristics that regulate spatial displacement of a tissue in one or more directions, comprising: a solid support comprised of at least two rigid attachment points for supporting a tissue. In some embodiments a first rigid attachment point is positioned at one end of the solid support and is connected to a compliant mechanism to allow desired displacements of a tissue relative to a second rigid attachment point. The spatial displacement may occur in at least a direction x, such that the tissue displacement is constrained in at least one direction other than x. The compliant mechanism may have a stiffness value in the direction of desired displacement that is lower than a stiffness value of the tissue in the direction of desired displacement and at least one of the rigid attachment points may have a stiffness value in the direction of desired displacement that is higher than the stiffness value of the tissue in the direction of desired displacement. In other aspects, this disclosure provides a device comprising; (i) a first attachment point; (ii) a second attachment point adjacent to the first attachment point, such that a tissue can interact with the first attachment point and the second attachment point; and (iii) a compliant mechanism arranged to produce stiffness characteristics having a first end and a second end; wherein the first end of the compliant mechanism is connected to a solid support and the second end of the complaint mechanism is connected to the second attachment point; and wherein the compliant mechanism has a lower degree of stiffness in one direction than at least one other direction.

[0082] An exemplary device is shown in the figures. For instance, FIG. 2A shows an example of a device having a solid support depicted as a base (18) which supports and connects other components. A compliant mechanism in the form of a flexure (10) is supported by the base through an optional mount (20) and is connected to the second attachment point in the form of a mobile pin (12). The connection between the flexure (10) and the mobile pin (12) in FIG. 2A is depicted as a direct connection. However, it is also possible for the compliant mechanism to be indirectly connected to the second attachment point through a linkage or connector. The base (18) also supports a first attachment point in the form of a ground pin (14). The first and second attachment points are positioned relative to one another such that a tissue can be wrapped around both points. When a force is applied to the system (i.e., the optical force produced by the optical fiber (22) depicted in FIG. 2B) to generate movement of the tissue the mobile pin (12) moves relative to the ground pin (14) which remains stationary, in the example depicted in FIG. 2A. However, in some embodiments both the first and second attachment points can be configured to move relative to one another.

[0083] Solid supports In some embodiments, the device comprises a solid support, for example a base (18) comprised of at least two attachment points (e.g., at least two rigid attachment points (4) and (6)). In some embodiments, the solid support is connected to a first end of a compliant mechanism (8). A solid support is a rigid or semi-rigid structure that provides stability, reinforcement, and / or a foundation for other components or structures. The solid support may be made of any biocompatible material that exhibits low viscosity, low stress relaxation, and / or low creep. Non-limiting examples of biocompatible materials include metals (e.g., titanium, stainless steel, zirconium, gold, and platinum), ceramics (e.g., aluminum oxide, zirconium dioxide, hydroxyapatite, and titanium dioxide), and biocompatible polymers. In some embodiments, the biocompatible polymer has a modulus larger than 0.1 gigapascals (GPa). Non-limiting examples of suitable biocompatible polymers include, without limitation, butyl rubber, cellulase acetate butyrate, cellulose acetate propionate, polybutylene, poly vinylidene chloride, nylon 11, high- density polyethylene, polytetrafluoroethylene, polyvinyl acetate, perfluorinated alkoxy, cellulose acetate, cellulose nitrate, nylon 46, polyvinylidene fluoride, nylon 6, polymethylpentene, polyhydroxybutyrate, polytrifluorochloroethylene, epoxy resin, ethylene tetrafluoroethylene, ethylene chlorotrifluoroethylene, nylon 66, high-impact polystyrene, nylon 612, polystyrene, acrylonitrile butadiene-styrene, polybutadiene, polyvinyl chloride, polycarbonate, polyethersulfone, chlorinated polyvinyl chloride, polyphenylene oxide, polysulfone, polybutylene terephthalate, polyetherimide, polyoxymethylene, polyethylene terephthalate, polymethyl methacrylate, natural rubber, casein formaldehyde, polyamide-imide, polyether ketone, polyvinyl fluoride, polyethylene naphthalate, unsaturated polyester, polybenzeneimidazole, and melamine formaldehyde. Other biocompatible materials exhibiting low viscosity, low stress relaxation, and / or low creep are contemplated. In some embodiments, the solid support is a base. In some embodiments, the base is attached to a first attachment point and a compliant mechanism. In some embodiments, a solid support (e.g., a base) comprising at least two rigid attachment points are fabricated from a single, uniform material (i.e., a monolithic device). In some embodiments, the solid support (e.g., a base) and at least two rigid attachment points are fabricated separately (i.e., a modular device). When fabricated separately, the components (e.g., the solid support, the at least two rigid attachment points) may be connected by any suitable means, including adhesive bonding (e.g., glue, epoxy, cyanoacrylate, polyurethane, acrylic) or mechanical fastening (e.g., screws, bolts, nuts, rivets, clips, snaps, loops). These methods may also be used to connect other components of this device (e.g., the compliant mechanism) to any other component of this device (e.g., an attachment point, a solid support). Advantages of such a modular device include, without limitation, the ability to vary the geometry of the device, the stiffness characteristics of each component, and the resolution of the motion control and displacement ratio.

[0084] Attachment points

[0085] An attachment point refers to a feature or a location where another object can be connected. Attachment points of the present disclosure can be made of any biocompatible material, such as those disclosed above. Exemplary attachment points are shown in the figures. For instance, FIG. IB is a schematic depicting a first (4) and second (6) attachment point, positioned relative to one another and with a tissue (2) wrapped around the attachment points. The second attachment point (6) is moved relative to the first attachment point (4). For instance, when the second attachment point (6) is moved a distance of xpbetween states A and B the distance that the tissue (2) is stretched is modified accordingly. In some embodiments, the attachment point is a rigid attachment point. In some embodiments, the attachment point is a pillar, a column, or a pin. In some embodiments, the attachment point is a pillar, such as a rigid pillar. Other suitable attachment points can be appreciated by those skilled in the art.

[0086] In some embodiments, the attachment point has a stiffness value in one or more directions that is higher than the stiffness value of a tissue in the one or more directions. A stiffness value refers to a measure of the resistance of a material or structure to deformation when subjected to a force. In some embodiments, the attachment point has a stiffness value in one or more directions that is 100-fold or more than the stiffness value of the compliant mechanism in the one or more directions. In some embodiments, the attachment point has a stiffness value in one or more directions that is 100-fold or more than the stiffness value of the tissue in the one or more directions. For example, the attachment point may have a stiffness value in one or more directions that is 100-fold, 125-fold, 150-fold, 175-fold, 175-fold, 200-fold, 225-fold, 250-fold, 275-fold, 300-fold, or more than the stiffness value of the compliant mechanism in the one or more directions and the stiffness value of the tissue in the one or more directions. In some embodiments, at least one of the rigid attachment points has a stiffness value in one or more direction that is higher than a stiffness value of a tissue in the one or more directions (e.g., having a stiffness ratio of 100 or more). In some embodiments, the attachment points (e.g., the rigid attachment points) have a stiffness value in one or more directions that exceeds the stiffness value of the tissue in the one or more directions.

[0087] In some embodiments, a device of the present disclosure comprises at least two attachment points (e.g., a first attachment point and a second attachment point). In some embodiments, a device of the present disclosure comprises a first attachment point and a second attachment point. In some embodiments, the at least two attachment points are the same (e.g., having the same stiffness characteristics). In some embodiments, the at least two attachment points are different (e.g., having different stiffness characteristics). In some embodiments, the first attachment point is adjacent to the second attachment point. In some embodiments, the first attachment point is adjacent to the second attachment point such that a tissue can interact with the first attachment point and the second attachment point. In some embodiments, a tissue is contacted with the first attachment point and the second attachment point. In some embodiments, a tissue is attached to the first attachment point and the second attachment point. Methods of attaching tissues to attachment points are known in the art and may vary depending on the tissue type, substrate material, desired strength of attachment, and intended applications. Non-limiting methods of contacting tissues to attachment points include physical methods (e.g., mechanical anchoring using pins, staples or sutures or vacuum-assisted techniques such as vacuum pressure), chemical methods (e.g., surface functionalization, such as with collagen, fibronectin, or other extracellular matrix proteins, crosslinking using agents such as glutaraldehyde or photochemical crosslinkers, or bioadhesives such as cyanoacrylate-based adhesives or fibrin glue), or biological methods (e.g., cell seeding onto a substrate). In some embodiments, cell seeding can be used to fabricate tissues in vitro into a variety of three-dimensional shapes (c.g., a ring). In a non-limiting example, a tissue ring (c.g., a muscle tissue ring) may be fabricated by filling a mold (e.g., a ring mold) with a cell-gel suspension of cells (e.g., myoblasts). In some embodiments, a tissue is placed around the first attachment point and the second attachment point. An example of such a process is depicted in FIG. 1A.

[0088] In some embodiments, a device of the present disclosure comprises at least two attachment points (e.g., 2, 3, 4, 5, 6, or more attachment points). In some embodiments, the device comprises two attachment points. In some embodiments, the device further comprises 2-4 additional attachment points. In some embodiments, the device comprises three attachment points. In some embodiments, the device comprises four attachment points. In some embodiments, the device comprises five attachment points. In some embodiments, the device comprises six, seven, eight, nine or ten attachment points.

[0089] In some embodiments, the tissue is further contacted with the additional attachment points (e.g., the third, the fourth, the fifth, the sixth, the seventh, the eighth, the ninth, and / or the tenth attachment points). In some embodiments, the tissue is further attached to the additional attachment points (e.g., the third, the fourth, the fifth, the sixth, the seventh, the eighth, the ninth, or the tenth attachment points). In some embodiments, the tissue is further placed around the additional attachment points (e.g., the third, the fourth, the fifth, and / or the sixth attachment points).

[0090] Complian t me chan isms

[0091] In some embodiments, a device of the present disclosure comprises a compliant mechanism. A compliant mechanism is a structure that exhibits controlled, deterministic displacement, and is capable of deforming under load and returning to its original shape when the load is removed. It is typically designed to exhibit controlled flexibility or compliance in specific directions. The functioning of an exemplary compliant mechanism is depicted in FIGs. 1C and ID. The schematic of FIG. 1C shows an exemplary compliant mechanism and muscle modeled as springs of stiffness kf and km, respectively, that exert forces on the second attachment point (6) in response to the muscle actuation force, Fac. The diagram of FIG. ID demonstrates that the muscle actuation is counteracted by the force the muscle expends on compressing itself, Fmc, thus reducing the force the muscle exerts on the compliant mechanism (8) to Fme.

[0092] An exemplary compliant mechanism in the form of a flexure (10) is shown in FIGs. 2A, 2B, and 2C. In some embodiments, the compliant mechanism has a first end and a second end. In some embodiments, the first end of the compliant mechanism is connected to a solid support (18). In some embodiments, the second end of the compliant mechanism is connected to an attachment point (e.g., the second attachment point). In some embodiments, the first end of the compliant mechanism is connected to the base by a mount (20). Compliant mechanisms of the present disclosure can be made using any biocompatible material that exhibits low viscosity, low stress relaxation, and / or low creep. Non-limiting examples of biocompatible materials include metals (e.g., titanium, stainless steel, zirconium, gold, and platinum), ceramics (e.g., aluminum oxide, zirconium dioxide, hydroxyapatite, and titanium dioxide), and biocompatible polymers. Non-limiting examples of biocompatible polymers have been disclosed above. In some embodiments, the compliant mechanism comprises a linear elastic material (e.g., low-density polyethylene (LPDE)). In some embodiments, the compliant mechanism comprises LPDE. In some embodiments, the modulus of the linear elastic material is between 0.1 Gpa and 10 Gpa. In some embodiments, the stiffness of the linear elastic material is between 0.1 Gpa and 1 Gpa, 0.1 Gpa and 2 Gpa, 0.1 Gpa and 3 Gpa, 0.1 Gpa and 4 Gpa, 0.1 Gpa and 5 Gpa, 0.1 Gpa and 6 Gpa, 0.1 Gpa and 7 Gpa, 0.1 Gpa and 8 Gpa, 0.1 Gpa and 9 Gpa, 0.1 Gpa and 10 Gpa, 1 Gpa and 3 Gpa, 1 Gpa and 5 Gpa, 1 Gpa and 7 Gpa, 1 Gpa, and 10 Gpa, 2 Gpa and 4 Gpa, 2 Gpa and 6 Gpa, 2 Gpa and 8 Gpa, 2 Gpa and 10 Gpa, 4 Gpa and 8 Gpa, 4 Gpa and 10 Gpa, 5 Gpa and 10 Gpa, 6 Gpa and 10 Gpa, or 8 Gpa and 10 Gpa.

[0093] In some embodiments, the compliant mechanism has a stiffness value in one or more directions that is lower than a stiffness value of the tissue in the one or more directions. In some embodiments, the stiffness ratio (e.g., the stiffness of the linear elastic material in one or more directions, kf, relative to the stiffness of the tissue in the one or more directions, km) is between 0.01-100 (e.g., between 0.01 and 1, 0.01 and 5, 0.01 and 10, 0.01 and 25, 0.01 and 50, 0.01 and 75, 0.01 and 100, 0.1 and 1 , 0.1 and 5, 0.1 and 10, 0.1 and 25, 0.1 and 50, 0.1 and 75, 0.1 and 100, fl and 5, 1 and 10, 1 and 25, 1 and 50, 1 and 75, 1 and 100, 5 and 10, 5 and 25, 5 and 50, 5 and 75, 5 and 100, 10 and 25, 10 and 50, 10 and 75, 10 and 100, 25 and 50, 25 and 75, 25 and 100, 50 and 75, 50 and 100, or 75 and 100). In some embodiments, when the compliant mechanism has a stiffness value in one or more directions that is lower than the stiffness value of the tissue in the one or more directions, displacement is maximized. In some embodiments, when the compliant mechanism has a stiffness value in one or more directions that is lower than the stiffness value of the tissue that correlates to the same direction of motion as the one or more directions, displacement is maximized. In some embodiments, the stiffness ratio is lower than 1 (e.g., 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, or less). In some embodiments, the compliant mechanism has a stiffness value in one or more directions that is about the same as a stiffness value of the tissue in the one or more directions (e.g., kf / km ~ 1). In some embodiments, when the compliant mechanism has a stiffness value in one or more directions that is about the same as the stiffness value of the tissue in the one or more directions, work output is maximized. In some embodiments, when the compliant mechanism has a stiffness value in one or more directions that is about the same as the stiffness value of the tissue that correlates to the same direction of motion as the one or more directions, work output is maximized. In some embodiments, the compliant mechanism has a stiffness value in one or more directions that is higher than a stiffness value of the tissue in the one or more directions (e.g., kf / km» 1). In some embodiments, the stiffness ratio is as high as 100 or more (e.g., 100, 125, 150, 175, 200, 225, 250, 275, 300, or more). In some embodiments, when the compliant mechanism has a stiffness value in one or more directions that is greater than the stiffness value of the tissue in the one or more directions, force output is maximized. In some embodiments, when the compliant mechanism has a stiffness value in one or more directions that is greater than the stiffness value of the tissue that correlates to the same direction of motion as the one or more directions, force output is maximized.

[0094] In some embodiments the degree of stiffness of the device can be modified. For instance, the compliant mechanism of the device comprises more than one functional beam which collectively provide a stiffness value. The beam or related components regulating the beam may be manipulated to alter the stiffness value of the compliant mechanism. The stiffness value may be reduced for example by reducing the number of functional beams, which can be achieved, for instance, by physically disrupting the body of the beams or their structural connections. As a nonlimiting example, the compliant mechanism may consist of five individual beams of varying beam thicknesses and / or lengths. One or more of the beams may be cut or the connections between beams or with the device may be cut to alter the overall stiffness of the compliant mechanism. Additional cuts or alterations to the beams may be made to further alter the stiffness. As shown in FIG. 13, the compliant mechanism (flexure bank) is coupled to a sensing flexure such that when the muscle pulls on the mounting mobile pin (12), the sensing flexure and flexure bank displace by the same distance. As the beams of the flexure bank are altered the stiffness is adjusted. As one example the spine of the flexure bank can be cut to remove one beam from the stiffness system. A study was done to assess the effect of altered stiffness values. The results are shown in Table 1 .

[0095] Table 1 Stiffness values of sensing assembly (sensing flexure and flexure bank).

[0096] Numberof Beams in Predicted Flexure Predicted System Predicted Predicted

[0097] Bank Bank Stiffness Stiffness Displacement Force

[0098] 5 125 N / m 193 N / m 9.61 pm 1.85 mN

[0099] 4 30.5 N / m 67.9 N / m 23.2 pm 1.58 mN

[0100] 3 13.8 N / m 37.5 N / m 35.4 pm 1.33 mN

[0101] 2 7.84 N / m 23.7 N / m 46.4 pm 1.10 mN

[0102] 1 7.84 N / m 15.9 N / m 56.3 pm 0.900 mN

[0103] 0 - 8.06 N / m 71.8 pm 0.589 mN

[0104] The flexure bank is the component that provides different stiffnesses for the muscle rings or other tissue to pull against. The exemplary flexure bank consists of five individual beams of varying beam thicknesses. During the experiment, each beam was cut in sequence until the muscle ring was only pulling against the stiffness of the sensing flexure itself.

[0105] In some embodiments, the compliant mechanism is a flexure. A flexure is a mechanical component or structure constructed and arranged to bend, flex, or shift in response to applied forces, while maintaining its structural integrity. Other compliant mechanisms similar to flexures include but are not limited to springs (device that stores mechanical energy and release it when deformed, allowing for controlled movement or force absorption), hinges (device that provides rotational movement around a fixed axis, allowing for controlled rotation or swinging motion), or bellows (device that is an expandable and collapsible accordion-like structure). In some embodiments, the compliant mechanism has a rectilinear topology. A rectilinear topology is a layout where components or elements are arranged in straight lines or along orthogonal axes (i.e., perpendicular). In some embodiments, the rectilinear topology comprises multiple parallel beams conjugated in series. In some embodiments, the rectilinear topology comprises between 2 and 50 parallel beams. In some embodiments, the rectilinear topology comprises 2-40, 2-30, 2- 20, 2-10, 4-30, 4-20, 4-15, 4-10, 5-30, 5-20, 5-15, or 5-10 parallel beams. Modifications to the geometry, dimensions, and spacing of the parallel beams are within the scope of this disclosure, and may be used to achieve desired characteristics using methods known to those skilled in the art without undue experimentation. In some embodiments, the compliant mechanism has non- rectilinear topology.

[0106] In some embodiments, the compliant mechanism has a low stiffness in one or more directions and / or orientations (e.g., x, y, z 0X, 0y, and / or 0Z). In some embodiments, the compliant mechanism has a high stiffness in one or more directions and / or orientations (e.g., x, y, z, 0X, 0y, and / or 0Z). In some embodiments, the compliant mechanism has low stiffness in one direction (e.g., x direction) and a high stiffness in two directions (e.g., y and z directions). In some embodiments, the compliant mechanism has low stiffness in one direction (e.g., x direction) and a high stiffness in all other directions and orientations (e.g., y, z, 0X, 0y, and 0Z). The skilled person can appreciate that the geometry and topology of the compliant mechanism enables controlled relative movement in all six degrees of freedom (e.g., x, y, z, 0X, 0y, and / or 0Z). In some embodiments, the geometry and topology of the compliant mechanism restricts relative movement to the tissue’s longitudinal axis (e.g., x direction). In some embodiments, the compliant mechanism has a lower degree of stiffness in one direction than in all other directions. In some embodiments, the tissue displacement is constrained in all directions other than x.

[0107] Adjustment mechanisms In some embodiments, an attachment point (e.g., a first attachment point) is attached to a solid support (e.g., a base) by an adjustment mechanism. An adjustment mechanism is a structure designed to facilitate the modification of specific properties, dimensions, or configurations to achieve a desired outcome. For example, in some embodiments, an adjustment mechanism of the present disclosure may allow the position of an attachment point (e.g., a first attachment point) to be adjusted in at least one direction or orientation (e.g., x, y, z, 0X, 0y, and / or 0Z) relative to another attachment point (e.g., a second attachment point). In some embodiments, the adjustment mechanism allows the position of an attachment point (e.g., a first attachment point) to be adjusted in one direction or orientation (e.g., x, y, z, 0X, 0y, or 0Z) relative to another attachment point (e.g., the first attachment point). In some embodiments, the adjustment mechanism allows the position of an attachment point (e.g., the second attachment point) to be restricted to one direction or orientation (e.g., x, y, z, 0X, 0y, or 0Z) relative to another attachment point (e.g., the first attachment point). In some embodiments, the adjustment mechanism is in a fixed position. Non-limiting examples of adjustment mechanisms include cams, wedges, screws, levers, and actuators. An exemplary adjustment mechanism is shown in FIG. 2A in the form of a cam (16). The cam, which also may be referred to as a tensioner may be used to account for the variation in length between muscle ring specimens. Using an eccentric cam and a flexure that constrains movement to occur only in one direction, the distance between the muscle mounting pins can be varied along the x-axis. This component controls the grounded pin, which remains stationary as the muscle rings pull against the mobile pin on the sensing flexure. In some embodiments the flexure and cam may be made from Invar 36. The grounded pin may be mounted to an LDPE component with a press-fit 1 mm pin as is the mounting component on the sensing flexure assembly.

[0108] In some embodiments, an adjustment mechanism is an actuator. An actuator is a component or mechanism responsible for generating controlled movement or action on one or more components or parts of a device. Actuators associated with nano- or micro-devices are known in the ail. Such devices play an important role, operating on the scale of nano or micrometers in manipulating and controlling these types of devices. Some non-limiting examples of actuators include piezoelectric actuators, electrostatic actuators, thermal actuators, and magnetic actuators. Piezoelectric actuators arc transducers that convert electrical energy into a mechanical displacement or stress based on a piezoelectric effect, allowing for precise movement or manipulation at the nanoscale. Electrostatic actuators operate based on the attraction or repulsion between charged components, enabling controlled movement or positioning of nanoscale elements. They are designed to simulate the pressure response of condenser microphones. Oscillating electrostatic force is exerted in front of the microphone diaphragm, providing the consequent response of the microphone. Thermal actuators convert a temperature change into a mechanical force to induce movement or actuation. Magnetic actuators use magnetic effects to generate forces to induce motion or alignment of components. In some embodiments, the actuator is configured to regulate spatial displacement of one or more components of the device. Other adjustment mechanisms are contemplated.

[0109] In some embodiments, the adjustment mechanism enables the distance between the at least two attachment points to be adjusted to match the passive length of a tissue. In some embodiments, the adjustment mechanism enables the distance between the at least two attachment points to be adjusted such that a tissue is optimally tensioned (e.g., greater than the passive length of the tissue) between the two or more attachment points. The optimal tension of a tissue refers to the ideal level of tension or stretch that maximizes a tissue’s function, mechanical properties, and / or performance. Those skilled in the ail can appreciate that the optimal tension of a tissue varies based on a number of external factors, including tissue type, biological factors (e.g., presence or absence of genetic mutations), injury or trauma, mechanical stress (e.g., repeated stretching), and other environmental factors.

[0110] In some embodiments, this disclosure provides a method of assessing a tissue with a device disclosed herein, comprising: (i) contacting a first attachment point and a second attachment point of a device with a tissue; (ii) stimulating the tissue or device one or more times to produce a force; and, (iii) measuring a property associated with displacement of the second attachment point relative to the first attachment point each of the one or more times. Other aspects of this disclosure provide a method for evaluating effect of a biologically active substance on a tissue, comprising: (i) contacting a tissue with a biologically active substance; (ii) applying a force one or more times; and (iii) measuring a property associated with displacement. In some embodiments, the tissue is positioned on a device disclosed herein and in contact with at least two attachment points on the device. In some embodiments, the property associated with displacement provides information on the effect of the biologically active substance on the tissue.

[0111] In contrast to prior art devices, the methods for assessing and evaluating tissue using the devices disclosed herein are highly sensitive and reproducible. The disclosed devices enable the movement of tissue in highly regulated manner, such that the tissue may be moved in one or more directions using precise controls and regulation. The highly regulated movements allow for more precise measurements to be made when studying the tissue. As a result, the methods have important implications for research into mechanistic or functional properties of the tissues, as well as screening of putative therapeutic or biologically active substances.

[0112] Forces and methods of applying

[0113] In some embodiments, this disclosure provides a method of stimulating a tissue to produce a force. In some embodiments, this disclosure provides a method of stimulating a device to produce a force. In some embodiments, this disclosure provides a method of applying a force (e.g., a normal force or a shear force) to a device described herein. In some embodiments, a device is stimulated electrically, mechanically, thermally, or magnetically to produce a force. In some embodiments, a force is applied to a device described herein using an actuator (e.g., an actuator attached to an attachment point). In some embodiments, a tissue is stimulated chemically, electrically, mechanically, thermally, or magnetically to produce a force. In some embodiments, a tissue is stimulated electrically (e.g., using electrodes to deliver electrical pulses). In some embodiments, a tissue is simulated chemically (e.g., applying biological agents that stimulate contraction pathways). In some embodiments, a tissue is stimulated optically (e.g., using fiber optics or LEDs). In some embodiments, the force is a normal force (e.g., a tensile force or a compressive force). A tensile force stretches or elongates an object (e.g., a tissue) along a longitudinal axis (i.e. the length of the tissue). In some embodiments, a device is electrically, mechanically, thermally, or magnetically stimulated to produce a tensile force. In some embodiments, a tissue is chemically, electrically, mechanically, thermally, or magnetically stimulated to produce a tensile force. In some embodiments, the force is a contractile force. A contractile force generated in contractile tissues results in a change in the tissue such as tissue shortening. In some embodiments, a device is electrically, mechanically, thermally, or magnetically stimulated to produce a contractile force. In some embodiments, a tissue is chemically, electrically, mechanically, thermally, or magnetically stimulated to produce a contractile force. In some embodiments, the force is a compressive force. In some embodiments, the force is a shear force. In some embodiments, the force is a torsion force.

[0114] Those skilled in the art can appreciate that the force can by produced by a stimulus at any appropriate frequency, duty cycle, and / or stimulus intensity depending on the desired effect. In some embodiments, the force can produced by a frequency of stimulation between 0.1 Hz and 100 Hz (e.g., between 0.1 Hz and 1 Hz, 0.1 Hz and 2 Hz, 0.1 Hz and 5 Hz, 0.1 Hz and 10 Hz, 0.1 Hz and 20 Hz, 0.1 Hz and 40 Hz, 0.1 Hz and 60 Hz, 0.1 Hz and 80 Hz, 1 Hz and 2 Hz, 1 Hz and 5 Hz, 1 Hz and 10 Hz, 1 Hz and 20 Hz, 1 Hz and 40 Hz, 1 Hz and 60 Hz, 1 Hz and 80 Hz, 1 Hz and 100 Hz, 2 Hz and 5 Hz, 2 Hz and 10 Hz, 2 Hz and 40 Hz, 2 Hz and 60 Hz, 2 Hz and 80 Hz, 2 Hz and 100 Hz, 5 Hz and 10 Hz, 5 Hz and 20 Hz, 5 Hz and 40 Hz, 5 Hz and 60 Hz, 5 Hz and 80 Hz, 5 Hz and 100 Hz, 10 Hz and 20 Hz, 10 Hz and 40 Hz, 10 Hz and 60 Hz, 10 Hz and 80 Hz, 10 Hz and 100 Hz, 20 Hz and 40 Hz, 20 Hz and 60 Hz, 20 Hz and 80 Hz, 20 Hz and 100 Hz, 40 Hz and 60 Hz, 40 Hz and 80 Hz, 40 Hz and 100 Hz, 60 Hz and 80 Hz, 60 Hz and 100 Hz, or 80 Hz and 100 Hz). In some embodiments, the force can be produced by low-frequency stimulation. In some embodiments, the low-frequency stimulation is less than or equal to 1 Hz (e.g., 1 Hz, 0.9 Hz, 0.8 Hz, 0.7 Hz, 0.6 Hz, 0.5 Hz, 0.4 Hz, 0.3 Hz, 0.2 Hz, 0.1 Hz, or less). In some embodiments, the force can be produced by high frequency stimulation. In some embodiments, the high-frequency stimulation is greater than or equal to 2 Hz (e.g., 2 Hz, 3 Hz, 4 Hz, 5 Hz, 6 Hz, 7 Hz, 8 Hz, 9 Hz, 10 Hz, 20 Hz, 40 Hz, 60 Hz, 80 Hz, 100 Hz, or more). Sensing Mechanisms, Properties associated with displacement and methods of measuring

[0115] In some embodiments, this disclosure provides a method of measuring a property associated with displacement. In some embodiments, the property associated with displacement is displacement, speed, velocity, acceleration, or combinations thereof. In some embodiments, this disclosure provides a method of measuring displacement. In some embodiments, displacement can be measured by evaluating a change in tissue length. In some embodiments, displacement can be measured by evaluating a positional or orientational change of one attachment point (e.g., a first attachment point) to one or more other attachment points (e.g., a second attachment point). Displacement can be measured in any number of ways including, without limitation, image tracking and other image -based methods, interferometry, strain gauges, capacitive sensing, and inductive sensing, and provide insight into the mechanical properties of a tissue, such as its elasticity, stiffness, strength, and structural integrity. In some embodiments, the property associated with displacement is speed. In some embodiments, the property associated with displacement is velocity (e.g., displacement over time). The velocity of tissue displacement can provide insights into the inherent properties of a tissue (e.g., modulus, viscoelasticity). In some embodiments, the property associated with displacement is acceleration.

[0116] In some embodiments, a tissue or device is stimulated one time. In some embodiments, a tissue or device is repeatedly stimulated (e.g., two or more times). Repeated stimulation of a tissue or a device can provide insights into fatigue resistance (i.e., the ability to withstand repeated mechanical stress without failure or damage) . In some embodiments, measurements of force and properties associated with displacement can be integrated evaluate force dynamics. For example, a device described herein can be used to calculate response time between stimulus and contraction onset, contraction time required to reach peak displacement, time at peak displacement, and relaxation time to passive resting state to provide insights into the contractile dynamics of engineered muscle.

[0117] The device may be used with an external sensing mechanism, and / or in some embodiments, the device may include a built-in sensing mechanism. In some embodiments the external sensing system may be an optical detector. Optical sensing provides a non-invasive, flexible method to track changes in displacement. Tt may be to track tissue displacement, generating data that can then be extrapolated to force and other quantities via beam bending approximations. Optical sensing equipment is often separate from the device of the disclosure. Optical methods for sensing, although relatively easy to use, often require significant postprocessing to extract useful data and may have threshold levels of accuracy and resolution when used on their own, which may limit some types of tissue characterization. External sensors such as optical sensors can be used with the devices of the disclosure alone or in combination with built-in sensors, for optimizing the amount and types of data that can be captured using the device.

[0118] A built-in sensing mechanism is a sensor system that is integrated into the device. Built- in sensors may be used alone or together with an external sensor. Built-in sensing mechanisms have many advantages when used with the devices disclosed herein. For instance, the inherent portability and miniaturization of the device may be enhanced such that the device is easily adapted for use with standard cell culture equipment such as multi- well plates. Another advantage of built-in sensors is the ability to capture low-stroke, high force measurements from tissue such as muscle, which are not achievable with optical systems. Built-in sensors can also make the use of a high-resolution, high-speed cameras to capture video data.

[0119] In some embodiments the built-in sensor is a strain gauge sensor. A strain gauge is a transducer that converts strain into a change in resistance. When connected in a Wheatstone bridge configuration, this resistance correlates to a detectable change in voltage. Strain gauges also typically have an amplification factor, called a gauge factor, that may be considered during selection. Strain gauges take advantage of the piezoresistive effect of materials to transduce a signal. This is due to two components: the geometric component and resistive component. The former is more prominent in metal gauges, where changes in cross-section and length affect the component’s resistance. Semiconductor gauges operate more on the subatomic level, where resistance changes are largely due to the change in atomic bonds. Thus, there are two types of materials commonly used for strain gauges: metal foil and semiconductor silicon. Metal gauges tend to have a lower gauge factor but are less sensitive to temperature changes. Semiconductor gauges have higher gauge factors (>100) but are more sensitive to changes in temperature. Another parameter which may be considered when selecting a strain gauge is the bridge configuration. The basic Wheatstone bridge circuit reads the voltage across two parallel arms of series resistors. When all elements have equal resistance, the output voltage is zero. When one resistance changes, the output voltage changes in proportion to that resistance change. Depending on the sensitivity desired, up to all four of the resistors in the Wheatstone bridge can be replaced with strain gauges. The positioning of these gauges in some arrangements take advantage of geometric material response to account for temperature change and Poisson effects. In selecting what type of strain gauge to use for this device, the material, gauge factor, and bridge configuration may be considered in tandem to achieve different functional requirements.

[0120] In some embodiments the sensing mechanism when built into the device is attached to the compliant mechanism. This subsystem may be referred to as a sensing flexure and includes a compliant mechanism, or flexure element, to which strain gauges are mounted. An exemplary device having a sensing flexure is shown in FIG. 12. The device is composed of four main components: the sensing flexure, the flexure bank (which is the flexure component of the compliant mechanism), the tensioner (also referred to as an actuator), and the frame or support. Unlike prior art devices, this device is not submerged in a petri dish alongside the muscle rings. Only two components interface directly with the muscles in the media, the two attachment pins. The muscle contraction is constrained to move along an axis aligned with the deflection of the sensing flexure, thereby maximizing its measured force output. By keeping the delicate sensors separate from the biological materials, this device does not require heat sterilization methods. The components that are required to be sterile are also low-cost and can be easily replaced throughout the lifetime of the equipment. In the shown device, the sensing flexure and flexure bank were made from low-density polyethylene. The tensioner and frame were made from Invar 36 to minimize any thermal errors .

[0121] The depicted assembly is composed of a straight beam with four semiconductor strain gauges, two on each end on opposite sides. This exemplary design was built and tested, but many alternative configurations and arrangements of beams and strain gauges are within the scope of the disclosure. The four exemplary gauges form two half-bridges, providing redundancy for data collection and thermal error compensation.

[0122] The exemplary device depicted in FIG. 12 was mounted via three 2.5 mm diameter pins which provide a press-fit between the flexure and the mount. One end of the flexure has a pinslot fit to avoid over-constraint and allow for thermal growth without warping the remainder of the beam. Channels in the mounting plate secured the lead wires connected to the semiconductor gauges. A rigid 2 mm diameter pin runs through the center of the sensing flexure. One end holds the mobile mounting pin for the muscle ring. When looped around this pin and the grounded pin (which is a part of the tensioner), the muscle displaces the mobile pin which translates into a signal transmitted by the strain gauges. The other end of this pin attaches to the flexure bank. When connected in parallel with the flexure bank, the same displacement is applied to both components. The sensing flexure assembly was made from low-density polyethylene. The strain gauges used in the exemplary sensing flexure assembly were unbacked gauges manufactured by Kulite, code ADP-250-220, made from D-type mono-crystalline silicon. The gauges have welded gold alloy leads, are robust under high temperature operation and have been shown to be accurate up to 5,000 microstrain in tension.

[0123] Exemplary Flexure Devices: Variable Stiffness Muscle Meters

[0124] In some embodiments the device of the disclosure is designed for use with high throughput tissue culture equipment such as multi-well plates. An exemplary design of the device in miniaturized form and / or with the provision of variable stiffness is provided herein. This exemplary design of the device is re I erred to in some embodiments as a variable stiffness muscle meter and is depicted in FIGs 8-11.

[0125] The variable stiffness muscle meter architecture possesses axisymmetric characteristics and is arranged for optimal fits within the circular footprint of a multi-well plate. The muscle (34) is mounted on pins MM1 (12, mobile pin) and MM2 (14, ground or fixed pin) as shown in FIG. 8A and 8C. Once mounted, the muscle may be (i) tensioned and / or (ii) subjected to different mounting stiffness. Those two functions are provided by the tensioning elements (36) shown on the top half of FIG. 8B and the variable stiffness elements (38) shown on the bottom half of FIG. 8B.

[0126] The tensioning mechanism is comprised of a tensioning arm (40) connected to a central point (42) that is mechanically grounded (does not move) by a rotary tensioning flexure bearing (TFB) (48). An actuator interfaces with the tensioning arm (40) at the tensioning actuation point (TAP) (50, shown in FIB. 8C). When actuated, the tensioning arm (40) is guided in rotation around the central point (42) by the rotary flexure bearing. This bearing ensures nominal rotary motion of the tensioning arm around the central point. This rotation, 9 in FIG. 8B, causes the upper attachment point for the muscle to move, thereby inducing a change in the spacing, 8 in FIG. 8B, of the two muscle attachment pins, MM1 (12) and MM2 (14) shown in FIG. 8C.

[0127] The variable stiffness mechanism is further depicted in FIGs. 8C-8D. Variable stiffness is achieved via interaction between the variable stiffness arm (38) and the variable stiffness flexure stage (VSFS) (52). The variable stiffness arm (38) is designed such that its cross section may vary along its length. The VSFS (52) is connected to the grounded central point (42) via the variable stiffness flexure bearing (VSFB) (54). The VSFS (52) and variable stiffness arm (38) exist as separate components, that is there is a small radial gap between them which is to be closed by a small preload during service. An actuator interfaces with the VSFS (52) at the variable stiffness actuator point (VSAP) (56). When actuated, the VSFS (52) travels nominally in an arcuate motion about the central point (42). This motion is specifically designed to track with the inner, adjacent surface of the variable stiffness arm.

[0128] When the VSFS (52) is actuated at different points along its arcuate motion, it makes contact with the inner, adjacent surface of the variable stiffness arm at different points. This is shown for example in FIG. 8D, where an actuation force causes the VSFS (52) to move through and angle p. This changes the location of the contact point, VSCP (41), between the VSFS and the variable stiffness arm (38), thereby changing the amount of the arm that will comply when the muscle pulls at the lower muscle attachment pin, MM2 (14). The cross section of the variable stiffness arm can be tailored to change along its length so that the effective stiffness felt by the muscle at MM2 (14) changes markedly with small actuated changes in p. In some embodiments the device comprises one or more actuators. For instance, exemplary devices having two actuators or one actuator arc shown in FIGs. 9 and 10 respectively. It is not uncommon for precision motors to have two degrees of freedom, for example two shafts that come out of one motor housing and which rotate independent of each other, which is essentially two motors within one motor housing. A conceptual cross section in Fig. 9 shows how shafts A and B, may be connected to the actuation points for the tensioning arm (TAP) (50) and the variable stiffness arm (VSAP) (56). This arrangement enables the tissue, such as muscle, and the pins (12) and (14) to be submerged within the well plate (60) while preventing contact between the mechanical components and the contained liquid.

[0129] In the exemplary embodiment depicting one actuator, the actuator (62) is used to position the VSFS (52) and the tensioning arm as shown in FIG. 10 The link coming off the single motor shaft is capable of swinging an arc (64) (see internal arc in FIG. 11) that enables it to make contact with either the rotary bearing for the tensioning arm (38) or the VSFS (52). The link is therefore able to push / actuate each rotary bearing towards position A or B as shown in FIG. 11. At positions A and B, there exists a means of affixing the respective flexure stage in that position. For example, a permanent magnet could be located at positions A and B so that it interacts with ferromagnetic material on each flexure bearing. When the ferromagnetic material on the flexure stages comes in close proximity with the permanent magnet, the interaction will fixate the flexure bearing in place. This scheme enables the motor to push the tensioning arm to a fixed point at A, release it and then rotate through the arc in FIG. 11 to then push the VSFS to point B .

[0130] This scheme depicted in FIG. 11 may be adapted so that there are multiple magnet locations that enable the motor to push the elements to more than one discrete position as they move towards points A or B. The positions may be undone by use of electromagnets located adjacent to the permanent magnets. When energized, the fields from the electromagnet cancel out the magnetic fields from the permanent magnet(s), thereby neutralizing their holding force. The rotary bearings would then spring back to the original positions / orientations. An alternative design for accomplishing the same function involves integrating a pushpush locking mechanism into positions A and B. Push-push mechanisms arc commonly used on mechanical devices (i.e., cabinet doors, involving pushing in on the door and to releases and close the door). The push-push mechanisms of the device of the disclosure may attain more than two states (e.g. extended and retracted in for instance a ball point pen analogy). Thus, a push- push-push-push mechanism could be integrated at point A, enabling 4 highly repeatable and discrete positions for adjusting tension. The same could be done at point B. The number of discrete positions and the spacing between them can be designed to provide the requisite resolution in tensioning and to provide discrete variable stiffness states.

[0131] Multi-well plate devices:

[0132] The devices of the disclosure in some embodiments are useful with microplates, such as multi-well plates. A multi-well plate may be, for instance, a 6, 12, 24, 48, 96, or 384 well plate, which is typically comprised of a base (30) that supports the wells (32), and a separate lid (33) which can cover, partially or fully, the plate. In some embodiments the device of the disclosure, partially or fully, may be attached to a bottom surface of the lid (33) or otherwise positioned at the bottom surface of the lid, as shown in FIG. 7. When the lid (33) is closed the pins or attachment points (12) and (14) are inserted into the well, such that they are in contact with the tissue and media. In some embodiments the flexure may be physically attached to the lid. In other embodiments, it is positioned adjacent to the lid but is not physically attached. In some embodiments the device forms a part of the lid.

[0133] Methods of screening

[0134] As noted above, a device provided herein can be used in a method for evaluating effect of biologically active substances. In some embodiments, a device provided herein can be used as a platform for drug development. Thus, without limitation, a device described herein can be used to screen for off-target effects, to identify agents with a desired effect on a tissue, to assess the potency and efficacy of an agent on a tissue, to characterize pharmacokinetics and pharmacodynamics. In some embodiments, tissue rings (e.g., skeletal muscle rings) fabricated using engineered cell populations (e.g., having characteristics of a healthy, diseased, or disordered cells) in contact with a device provided herein can be contacted with a biologically active substance, and the effect of the biologically active substance on the tissue (e.g., activity, physiology, function, or other mechanical properties) can be assessed (e.g., relative to the tissue ring that is not contacted with the biologically active substance). Alternatively, the cells may be derived from a patient with a known disease or disorder. In such an instance, methods provided herein permit screening to identify biologically active substances that can correct or ameliorate the defect or anomalous function. In other embodiments, a device of the present disclosure can be used to compare the activity, physiology, function, or other mechanical properties of one or more tissues (e.g., a healthy tissue and a diseased tissue) in the presence of absence of a biologically active substance. Other tissue- specific properties are contemplated and can be determined by those skilled in the art. For example, parameters to evaluate muscle tissue may include, but are not limited to, action potential frequency, duration of activation, threshold of activation, contractile force, velocity of contraction, duration of contraction, peak systolic stress, frequency of contraction, peak force, time to peak force, and viscoelasticity.

[0135] In a non-limiting example, a drug screening method utilizing a device provided herein could be as follows: A microplate (e.g., a 6, 12, 24, 48, 96, or 384 well plate) can be used to evaluate the effect of a biologically active agent on one or more properties of a tissue (e.g., work output, contractile displacement, force output) by differentially stimulating the tissue or device (e.g., by altering the type, magnitude, frequency, duration, and / or direction of the stimulus) to each row of the microplate. In another non-limiting example, a drug screening method utilizing a device provided herein could be as follows: A microplate (e.g., a 6, 12, 24, 48, 96, or 384 well plate) can be used to evaluate the effect of multiple biologically active agents on one or more properties of a tissue (e.g., work output, contractile displacement, force output) by applying a different biologically active agent to each row of the microplate.

[0136] Tissues Aspects of this disclosure are directed to evaluating a tissue using a device of the present disclosure. In some embodiments, the tissue is a contractile tissue. A contractile tissue is a type of tissue that has the ability to contract in response to a stimulus. In some embodiments, the contractile tissue is a muscle, such as skeletal muscle, cardiac muscle, or smooth muscle. In some embodiments, the tissue is a non-contractile tissue. Non-limiting examples of non- contractile tissues include connective tissue (e.g., tendons, ligaments, cartilage, or bone), epithelial tissue (e.g., skin or blood vessels), and nervous tissue (e.g., neurons, glia, or ganglion).

[0137] In some embodiments, the tissue is a healthy tissue. In some embodiments, the tissue is a diseased tissue. A diseased tissue refers to an abnormal or pathologically altered tissue that deviates from its normal structure, function, or physiology due to infection, injury, inflammation, genetic mutations, and / or systemic disease. Diseased tissues may exhibit a range of structural, biochemical, and functional changes depending on the underlying cause and severity of the disease. In some embodiments, the muscle tissue is composed of myoblast. In some embodiments, the myoblasts are healthy myoblasts. In some embodiments, the myoblasts are diseased myoblasts. Diseased myoblasts may arise in various pathological conditions, such as muscular dystrophies (e.g., Duchenne Muscular Dystrophy or Becker Muscular Dystrophy), inflammatory myopathies (e.g., dermatomyositis, polymyositis, inclusion body myositis), muscle atrophy, such as atrophy resulting from disuse, aging, or systemic diseases (e.g., cachexia, sarcopenia), metabolic myopathies (e.g., glycogen storage disease), or acute or chronic muscle injury (e.g., strains, tears, contusions).

[0138] Biologically active substances

[0139] Aspects of this disclosure provide a method of evaluating the effect of a biologically active substance on a tissue. Biologically active substances are compounds or molecules that interact with biological molecules (e.g., proteins, enzymes, receptors, DNA) and influence physiological processes, cellular functions, and / or biochemical pathways. Biologically active substances of the present disclosure can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds (e.g., in the form of bacterial, fungal, plant, and animal extracts). In some embodiments, the synthetic or natural compounds can be modified through conventional chemical, physical, and biochemical means (e.g., to produce structural analogs, or to introduce mutations or chemical modifications). In some embodiments, a biologically active substance of the present disclosure is a pharmaceutical composition (e.g., comprising an active substance and one or more excipients).

[0140] In some embodiments, the biologically active substance is a small molecule (e.g., an organic or an inorganic molecule), a protein, a lipid, or a nucleic acid (e.g., DNA or RNA). In some embodiments, the biologically active substance is a pharmacologically active drug. In some embodiments, the biologically active substance is a gene therapy product. Non-limiting examples of biologically active substances antibiotics, antivirals, antifungals, antiparasitics, antihistamines, hormones, steroids, neurotransmitters, anesthetics, antianginas, antiarrhythmics, antiasthmatic agents, antidiabetics, antihypertensives, immunomodulators, antibodies, such as monoclonal antibodies, sedatives, analgesics, vaccines, tranquilizers, RNA, DNA, peptides, proteins, and vasopressors. Other biologically active substances, known and unknown, are contemplated.

[0141] EXAMPLES

[0142] In order that the invention described herein may be more fully understood, the following examples arc set forth. The examples described in this application arc offered to illustrate the methods, compositions, and systems provided herein and are not to be construed in any way as limiting their scope.

[0143] Example 1. Enhancing and Decoding the Performance of Muscle Actuators with Compliant Mechanisms.

[0144] This Example demonstrates successful design of compliant mechanical elements, such as flexures, that enhance muscle contractile stroke to ~5X previously reported values and the decoding of contraction dynamics with high spatiotemporal resolution. The results illustrated in this Example show that combining rigid and flexible elements within a compliant mechanism (e.g., a flexure) enables outperformance of the sensitivity of gold standard elastomeric beam- based measurements of muscle contraction at both low- and high-frequency stimulation. This Example additionally demonstrates leveraging of compliant mechanisms to make quantitative comparisons of force, work, and power outputs in muscle actuators. Using complaint mechanisms disclosed herein, the inventors have surprisingly found a new observation of frequency-dependent fatigue in muscle and developed a novel method for tuning muscle contractile dynamics in a frequency-independent manner.

[0145] Previous efforts to characterize the performance of engineered tissues have almost exclusively relied on flexible pillar-based methods (as shown in FIG. 3B) to track contractile displacement and calculate force. In this technique, tissues are grown around compliant beams of measurable geometry and constitutive properties. The deformation of the beam in response to muscle contraction is recorded in videos, and sequential images are processed by manual or computer- associated visual tracking to output deflection as a function of time. Established principles of Euler-Bernoulli beam theory are then leveraged to convert deflection into contractile force. While beam-based force measurement is a well-established gold standard in this field, its accuracy is inherently limited by uncertainties in measuring the placement of muscle along the length of the beam, as well as the contact area between the muscle and pillar. Furthermore, the beams themselves are typically treated as linear elastic materials experiencing small deformations, despite the fact that they are generally fabricated from very compliant hydrogels and polymers (such as poly (ethylene glycol) diacrylate (PEGDA) and poly dimethyl siloxane (PDMS)) that are viscoelastic and experience large deformations and rotations in response to muscle contraction. The assumptions made by beam-based calculations of force thus significantly reduce the ability to accurately calculate forces, particularly in response to high- frequency stimulation. As muscle typically outputs its largest forces at its highest operating frequency (10+ Hz, termed tetanic contraction), this limitation poses a significant challenge.

[0146] To address this gap in the field, a few studies have explored the use of flexible electronic sensors to monitor the contractility of muscle. In the context of engineered cardiac muscle, for example, 2D and 3D bioelectronic interfaces have largely been used to dynamically monitor changes in electrical potential during contraction. A recent study focusing on engineered skeletal muscle showed that interfacing bioclcctronic strain sensors with tissues can also be used to extract quantitative measurements of forces in real-time. This technique was validated by demonstrating that strain sensor-based measurements of forces matched those generated by traditional pillar-based optical tracking at low frequency stimulation (< 1 Hz), and more reproducibly measured forces at higher frequency stimulation (> 2 Hz). While this study presented and validated one of the only non-beam-based techniques for monitoring contractile tissue forces, there were discrepancies in measurement impacted by the shape and contact area of the muscle tissue with the beam, suggesting the need for a novel beam-based method.

[0147] Force measurement using flexible electronics requires complicated and expensive multimaterial microfabrication techniques, and sensors must be uniquely designed and manufactured for each target application. Moreover, as such sensors must be wrapped around tissues during measurement, they restrict motion during contraction. This is a critical design flaw for interfacing with muscle, as it is a strain-limited actuator with a limited dynamic range of movement. Skeletal muscle, for example, is composed of aligned arrays of independent fibers which shorten in response to external electrical stimuli. Large motions are enabled by on / off control of contraction, which typically approaches -20% of original length in natural muscle and tissues derived from primary cells and -1% in engineered tissues derived from established cell lines. Sensor conformations that restrict the contraction of muscle fibers, therefore, severely impact their usefulness for real-time force measurement.

[0148] The devices disclosed herein overcome these difficulties and: 1) reproducibly monitor tissue forces with high precision without severely limiting muscle contraction; 2) are insensitive to changes in the geometry and placement of muscle tissues that are a natural consequence of using nonhomogeneous biological materials; and 3) can be easily and inexpensively manufactured. These devices enable more predictable and modular design of biohybrid machines. This Example describes the development of a simple and purely mechanical system that leverages compliant mechanisms, i.e., flexures to decode the dynamics of muscle contraction with high resolution in force and time. This technique is not dependent on muscle geometry or placement, and thus outperforms the robustness and sensitivity of existing beam-based techniques used to quantify tissue contractility. Compliant mechanisms, moreover, enable precise monitoring of the dynamics of shortening and relaxation throughout a muscle contraction pulse, rather than recording a single spike value of maximum force, as is the general standard in this field.

[0149] This Example additionally shows the design of exemplary compliant mechanisms optimized to enhance and decode the performance of skeletal muscle, the tissue that controls all voluntary movement in the body. The results of this Example demonstrate that compliant mechanisms disclosed herein outperform the sensitivity of traditional beam bending-based methods for quantifying muscle performance. Moreover, this Example demonstrates that compliant mechanisms described herein can be leveraged to maximize the contractile strain output of engineered skeletal muscle to ~5X previously reported values. This Example demonstrates robust characterization of muscle response time, contraction speed, and relaxation speed and showcases how performance dynamics, such as time at peak force, can be precisely tuned by modulating stimulation parameters in a non-physiological manner. This Example also compares frequency-dependent force, work, and power outputs, highlights a new observation of frequency-dependent fatigue in muscle, and enable precisely tuning the force output and endurance of engineered muscle actuators.

[0150] Results

[0151] Design, Fabrication, and Characterization of Compliant Mechanisms for Monitoring Muscle Fabrication of Engineered Skeletal Muscle

[0152] Skeletal muscle has many characteristics that make it highly desirable as an actuator for engineered machines. Native skeletal muscle has a hierarchical structure that enables scalability from the micro- to the macro-scale and outperforms most synthetic actuators in efficiency and mass / power ratio. In recent years, several efforts to reproduce these performance characteristics in engineered tissues fabricated from cell lines have yielded robust protocols for manufacturing contractile skeletal muscle in lab settings. However, these tissues largely do not match the contractile strain of native muscle, motivating the need to decode and enhance the performance of muscle tissues in vitro.

[0153] Most protocols for fabricating muscle rely on embedding myoblasts in natural hydrogels that mimic the physical and biochemical environment of the native extracellular matrix, and then differentiating them into multinucleated contractile muscle fibers. Engineered muscle was fabricated following established techniques by embedding murine myoblasts from the C2C12 cell line in a fibrin and Matrigel matrix, and culturing them in media designed to promote cell proliferation (FIG. 1A). As myoblast proliferate throughout the matrix, they exert traction forces on the surrounding gel to compact into a 3D tissue ring that can be transferred to a wide variety of physical support structures, showcasing the modularity of the ring design. Tissues were differentiated by altering the media composition (i.e., replacing fetal bovine serum with horse serum, and supplementing with human insulin-like growth factor 1 (IGF-1)) to trigger myoblast fusion into mature contractile multinucleated fibers.

[0154] Mathematical Model of Muscle -Compliant Mechanism Interaction

[0155] Compliant mechanisms are mechanical elements with tunable compliance along specific degrees of freedom of motion and can be modeled as elastic springs. An ideal compliant mechanism design can guide the movement of skeletal muscle along specific degrees of freedom, provide minimal resistance to contraction, and feature reliable geometric artifacts for automated optical measurements. Designing such a compliant mechanism first required modeling muscle in its passive and actuated states in both unconstrained and constrained environments. The effect of the muscle-compliant mechanism interaction was modeled by wrapping the muscle around two rigid pins, wherein one pin is immobile (first attachment point, (4)) and one pin is mobile (second attachment point (6)), but may only move in the x direction, aligned with the muscle fibers (FIG. IB). In a passive state, defined as State A, the muscle (2) is at rest and at its full length. Actuation of the muscle (2) (State B) allows unconstrained contraction of the tissue (2) to its maximum amount, defined as xp. If an external load is superimposed on the second attachment point i.e., mobile pin (6) via a compliant mechanism, such that is acts opposite to the muscle force (State C), actuation of the muscle will generate a smaller net movement, defined as Xi.

[0156] The compliant mechanism and muscle can be modeled as springs that exert forces on the second attachment point i.e., mobile pin (FIG. 1C). In the absence of compressing itself, the muscle would be able to exert Facon the external load (compliant mechanism with stiffness kf). The muscle must, however, exert force ( ,nf) to compress itself against its own stiffness, km(FIG. ID). Therefore, the amount of force the muscle can exert, F / ne, on an external load is lower than Fac. As the muscle contracts, the loss in force that it can exert is reduced linearly in proportion to the product of its stiffness and amount of compressive displacement. This behavior is shown in FIG. IE, which superimposes a plot of the linearly decreasing muscle exertion force, Fme, and linearly increasing compliant mechanism force, Ff.

[0157] As the compliant mechanism and muscle are unstrained at State A, and then undergo the same displacement, the intersection, (xi, Fi) of the two curves represents an equilibrium point. At this point, the muscle exertion force at a given x displacement is equal to the force required to displace the compliant mechanism by the same x displacement. The muscle exertion force may be mathematically modeled via Equation 1 between points (0, Fp) and (xp, 0). The displacement xprepresents the peak displacement the muscle may exhibit when unloaded and the force Fprepresents the peak force the muscle may exert. The peak force would be obtained if the muscle were actuated, and neither pin could move so the muscle was unable to compress itself. Equation 1

[0158] The stiffness of a compliant mechanism determines the intercept force and displacement, Xi and Fi, and the amount of work the muscle does upon the compliant mechanism. To design a compliant mechanism to achieve specific targets for displacement or work, the stiffness of the compliant mechanism must be defined parametrically as in Equation 2.

[0159] Equation 2

[0160] It is possible to target the intercepts via Equation 3.

[0161] The energy stored in the compliant mechanism may be obtained via the application of

[0162] Equations 4 and 5.

[0163] Equation 4

[0164] Where the constants obtained through integration are found to be 0 due to boundary conditions and therefore the stored energy in the compliant mechanism is:

[0165] Equation 5

[0166] The maximum stored energy in the compliant mechanism thus occurs at I / 2 xpper

[0167] Equation 6.

[0168] Equation 6

[0169] At this displacement, the maximum stored energy occurs when the stiffness of the compliant mechanism and muscle stiffness are equal, as represented in Equation 7.

[0170] Equation 7

[0171] At this stiffness, the maximum work that can be done on the compliant mechanism may be expressed as a function of the muscle’s peak force and peak displacement (Equation 8).

[0172] Equation 8

[0173] The total stored energy in the compression of the muscle and the straining of the compliant mechanism can be obtained via Equation 9. From inspection, the work done to strain the compliant mechanism, and the work the muscle docs to compress itself, must be equal.

[0174] Equation 9 One may select compliant mechanisms having other stiffness values to target other classes of behaviors. Stiffnesses that enable high stroke (kf / km<< ) arc useful to testing the capability of muscles, as demonstrated in this Example. By contrast, higher load stiffnesses that match (kf / km = 1) or exceed (kf / ktn>>1) natural muscle stiffness will increase work or force output, respectively, as may be desired for different soft robotics applications.

[0175] Manufacturing and Validation of Compliant Mechanisms

[0176] To characterize muscle contractile forces and dynamics with high resolution in space and time, compliant mechanisms tuned for high-stroke were manufactured, enabling ready visualization of single pulses and multiple pulses over time via high frame rate imaging. Muscle stiffness was calculated to be ~30 N / m using empirically measured values for modulus (Em), cross-sectional area (Am), and length (Lm) according to Equation 10, and thus a compliant mechanism stiffness of ~0.3 N / m (corresponding to kf / km= 0.01 <<1) was used for all subsequent experiments to maximize muscle strain. Equation 10

[0177] Compliant mechanisms were constructed from biocompatible low-density polyethylene (flexure (10) and base (18)) and Teflon (pins (12) and (14)) via precision milling. The compliant mechanism (e.g., flexure (10)) was mounted and grounded to the base via mount pins (20), and the muscle (2) was placed around the first (14) and second attachment (12) points (FIGs. 2A- 2B). The topology of the compliant mechanism (e.g., flexure (10)) was designed with several parallel beams conjugated in series such that the aggregate yields a low stiffness in the x direction while maintaining relatively high stiffness in the y and z directions. This strategy of low x stiffness value minimizes the load the muscle must pull with, thereby maximizing the energy available to actuate statically and dynamically. The high y and z stiffness values were selected so that the compliant mechanism guided the muscle displacement in a way that muscle displacements may be reliably measured optically and energy is not wasted on deformation in other directions. These characteristics were confirmed via finite element analysis modeling (FIG. 2C). The device also integrated cams to adjust the spacing between the ground and mobile pins to match the passive length of the muscle tissue. Specifically, the cams possessed eccentric features such that rotating them in their lightly press-fit bore made it possible to change pin spacing. Finally, at the frequency and speeds the muscle is being actuated (1-4 Hz), the drag force from the compliant mechanism moving in the nutrient media bath is estimated to be several orders of magnitude less than the muscle exertion force, thus permitting characterization of the muscle’s ability to deform in a guided way under its own influence.

[0178] Enhancing Muscle Contractile Strain with Compliant Mechanisms Enables Outperforming Precision of Beam-Based Techniques

[0179] Contraction of native muscle is typically triggered by an electrical signal from an upstream motor neuron that opens voltage-gated calcium ion channels in the muscle fiber membrane. Stimulation of differentiated muscle in vitro can thus be triggered by an electrical pulse, but this method of activation can often generate electrolysis of the media and negatively impact cell viability. Thus, a previously established optogenetic myoblast line expressing a lightgated calcium ion channel (ChR2[H134R]) was leveraged to non-invasively trigger muscle contraction in response to 470 nm blue light stimulation.

[0180] To confirm optically-triggered contractility and establish the maximum contractile strain that could be generated by muscles, videos of untethered free-floating muscle (2) in response to light stimulation at 1 Hz were recorded (FIG. 3A). Contraction of the same muscle (2) when placed on a traditional beam-based device (24) manufactured from PDMS (stiffness -20 N / m, as used in prior studies) and the custom compliant mechanisms (e.g., flexures (10)) were also recorded (FIGs. 3B-3C). Comparing the maximum contractile strain across these three modes of measurement for the same set of tissues showed that compliant mechanisms maximized the contractile output of engineered muscle and significantly outperformed beam-based systems (FIG. 3D). Surprisingly, a significant increase in measured strain on compliant mechanisms as compared to unconstrained muscle was noted, as the unconstrained case should represent the theoretical maximum contractile displacement of the tissue. This surprising observation was attributed to the fact that compliant mechanisms constrain the muscle’s degrees of freedom of motion to match the alignment of muscle fibers within the tissue, thus maximizing the measurable contractile strain. This explanation was corroborated by further noting the improved reproducibility of strain output on compliant mechanisms, as compared to the large standard deviation of measured strains for untethered muscle. Measurement reproducibility was also improved on compliant mechanisms as compared to beams, which was expected due to the fact that beam displacement is very sensitive to the exact placement location and contact area of the muscle. Given that compliant mechanisms both maximized contractile strain output and improved the reproducibility of measurement as compared to the current state-of-the-art, the compliant mechanisms were subsequently tested to determine whether they could be deployed as diagnostic tools to precisely distinguish differences between muscles.

[0181] A key concern of manufacturing living actuators is the need to account for batch-to-batch variability in the natural biological reagents that are key ingredients for fabricating mature muscle. While Matrigel provides critical biochemical cues for developing skeletal muscle that have yet to be matched by synthetic gel platforms, it is known for significant differences in composition between various formulations that generate uncertainty and lack of experimental reproducibility. Deploying muscle actuators in robotic applications thus requires new quality control methods that enable quantifying the impact of specific batches of biological reagents on tissue maturation and function. Muscle was generated from two versions of Matrigel (low growth factor (GF) and high GF) and the peak displacement at 1 Hz stimulation on flexible beams and compliant mechanisms was compared (FIGs. 3E-3F). Since the rigid posts of the compliant mechanism device were insensitive to muscle placement, differences in performance across samples could be attributed to variations across specific muscle tissues, rather than variations in how they were manually placed on the device. Moreover, as the compliant mechanism device contained clearly defined and trackable edges, tools for automated computational image tracking were leveraged to rapidly and accurately monitor displacement over time without the need for manual clicking-based tracking. As a result of these technique advantages, compliant mechanism devices could clearly resolve differences between muscles made from the two types of Matrigel with high statistical significance, whereas the relatively large inter-device measurement variability in pillar-based techniques did not enable distinguishing a significant difference. This served as a promising indication that this exemplary compliant mechanism platform enabled precise quantitative tracking of muscle displacement during contraction, enabling more reliable quality control than possible with established gold standard methods.

[0182] Monitoring and Modulating Contractile Dynamics

[0183] The majority of prior studies on engineering functional skeletal muscle report peak contractile displacement or force, but largely neglect studying the dynamics of muscle contraction and relaxation across a single pulse. While this can be attributed to the limited spatiotemporal resolution and reliability of pillar-based tracking, it indicates that there is a dearth of understanding of the contractile dynamics of engineered muscle. Leveraging the compliant mechanism-based system described above, the following were calculated: 1) response time between light stimulus and contraction onset; 2) contraction time required to reach peak displacement; 3) time at peak displacement; and 4) relaxation time to passive resting state. All initial measurements were made at 1 Hz stimulation with a 20% duty cycle (FIG. 4A). Average values of 48 milliseconds response time, 170 milliseconds contraction time, 78 milliseconds time at peak force, and 320 milliseconds relaxation time were observed (FIG. 4B), with the largest variability in contractile dynamics observed in relaxation time.

[0184] The relaxation dynamics of muscle play a critical role in the tissue’s frequency-dependent response, as higher-frequency stimulation may trigger subsequent pulses of muscle contraction before the muscle has fully returned to its passive state following a prior stimulus. In these cases, the dynamic range of motion is reduced, and tetanic twitch (i.e. sustained contraction that results in no net displacement) is observed in both natural and engineered muscle at high-frequency stimulation. In natural muscle, contraction and relaxation dynamics are primarily determined by the proportion of different muscle fiber types that are distributed in the muscle, ranging from slow twitch (Type 1) to fast twitch fibers (Types 2A, 2B, 2X). This can be attributed to the fact that the duty cycle of the muscle input stimulus is determined by the fixed dynamics of depolarization in upstream motor neurons. Previous studies of engineered skeletal muscle typically stimulate muscle with pulses in the range of 1-50 ms, attempting to match the dynamics of neuron stimulation, and thus report observing tetanus in the range of 10-40 Hz. While this may be relevant for studies with medical relevance, it is important to note that there is no inherent biological limitation on the duty cycles of stimuli used to activate muscle contraction. There is thus significant flexibility in tuning contraction dynamics, such as time spent at peak force, to the needs of specific robotics applications without changing muscle fiber types. However, no other studies have yet investigated whether tuning stimulation duty cycle can be used to modulate time spent at peak force and low-frequency induction of tetanus.

[0185] To investigate this phenomenon, muscle contraction in response to light stimulation at 1 and 4 Hz at 20, 50, and 80% duty cycle (DC) was tracked to observe changes in muscle displacement dynamics during twitch (FIG. 4C). Established elastomeric beam-based mathematical models for converting displacement data into force typically neglect viscoelastic response at high-frequency stimulation, and thus generate less accurate measurements of force output at higher frequencies. The compliant mechanism platform, which couples muscles to linear elastic springs of defined stiffness through rigid posts, mitigates the need to account for viscoelastic behavior of the flexural spring, thereby increasing the accuracy of high-frequency tracking. As muscle fiber type distribution within the tissue would not be impacted by the dynamics of an external pulsed stimulus, frequency-dependent or duty cycle-dependent differences in contraction speed were not expected nor observed. However, the time spent by muscles at peak displacement did vary significantly as a function of light pulse dynamics (FIG. 4D).

[0186] Further, by leveraging tunable duty cycles to precisely modulate time spent at peak contraction, it was shown that the muscle’s dynamic range of motion could be reduced by triggering subsequent pulses of contraction before the prior contraction / relaxation cycle had been completed. While the established understanding in the field of skeletal muscle biology is that the tissue’s dynamic range of motion can only be tuned by changing the frequency of stimulation, these results show that increasing duty cycle at the same frequency can also be used to modulate twitch duration. Moreover, the ability to induce tetanic contraction at 4 Hz (with an 80% DC), a significantly lower frequency than observed in previous studies of native muscle or engineered muscle tissues (10-40 Hz), was demonstrated by precisely tuning optical pulse dynamics (FIGs. 4E-4F). This phenomenon is particularly interesting as it enables, for the first time, decoupling the dynamic range of motion per twitch with the time spent at peak force.

[0187] Frequency-Dependent Force, Work, and Power Output

[0188] The force generated by the engineered muscle transferred to the compliant mechanism device can be calculated using measured values of maximum untethered displacement (x ), muscle tissue stiffness (kmand compliant mechanism stiffness (kf) (FIG. IE). Plotting forcedisplacement relationships for the untethered engineered muscles and compliant mechanism enabled visualization of the intersection of these lines, thus attaining a predicted value of displacement (x,) and force output (F,) for the biological actuators (FIG. 5A). Interestingly, the observed displacements exceeded predicted values (FIG. 5B), which was attributed to the fact that the measurements of maximum untethered displacement were derived from free-floating muscle with no constrained degrees of freedom of motion. As noted in FIG. 3D, compliant mechanisms enhance observable muscle strain by constraining the degrees of freedom of motion during contraction, and thus record significantly higher displacements (with smaller standard deviations) than unconstrained muscle. Since increased compliant mechanism displacement corresponds to increased force, it was calculated that the observed forces exceeded predicted values (FIG. 5C).

[0189] Plotting twitch force output as a function of time for several muscle actuators at 1 Hz and 4 Hz (20% DC) revealed that, as expected, the largest force change from baseline per twitch occurred at low frequency where the muscle could travel through its entire range of movement during a contraction / relaxation cycle (FIG. 5D). At higher frequency, as the dynamic range of motion is decreased, the change in force per twitch is likewise reduced (FIG. 5E). The total work done by the muscle per contraction cycle is equal to the sum of the work stored in the compliant mechanism and the work stored in the tissue compliance, as described in Equation 11. Equation 11

[0190] Plotting work per cycle reveals that the work generated by muscle per individual twitch is significantly higher at low frequency stimulation (FIG. 5F). However, normalizing work per unit time revealed that total power output is actually significantly increased at high frequency stimulation (FIG. 5G). These results corroborate prior observations of functional differences in muscle-powered machines actuated at different frequencies. For example, it was previously observed that the walking speed of a skeletal muscle-powered locomotive robot can be increased by increasing the stimulation frequency from 1 to 4 Hz, despite the fact that each “step” taken by the muscle is smaller at high-frequency stimulation. While prior studies did not attempt to provide an explanation for this observation, the results of this Example indicates that the observed improvements in functional performance could be attributed to increased power output at higher frequency stimulation, despite reduced dynamic range of motion.

[0191] Fatigue Response of Muscle Actuators

[0192] The compliant mechanism platform enabled stable longitudinal tracking of muscle over extended time periods. This stability was leveraged to monitor fatigue behavior of muscle tissues during chronic stimulation as no prior studies have conducted a systematic investigation of engineered muscle endurance in vitro. Muscle contraction was stimulated at 1 and 4 Hz (20% DC) for 30 minutes, and monitored to determine the difference in peak displacement at 0 min compared to peak displacement at 15 and 30 minutes. These experiments revealed no significant fatigue after 30 minutes of continuous activation at 1 Hz, but recorded a significant drop in performance at 4 Hz (FIGs. 6A-6B).

[0193] Plotting force-velocity relationships at 0, 15, and 30 minutes for 3 representative muscle tissues at 1 and 4 Hz showed that, in all cases, tissues returned to a passive zero velocity state in between cycles (data not shown). Interestingly, though the muscles did not demonstrate a significant drop in performance after 30 minutes of 1 Hz stimulation, they traversed slightly different force-velocity curves for each cycle, with variability in output appearing to increase with increasing duration of stimulation. These results indicates that, even when considering a single muscle actuator in isolation, the force output in response to an input stimulus is a function of the time history of that actuator. Overall, the fatigue study suggests that previous contraction cycles are a dynamic state variable that must be considered when predicting future performance of a living actuator.

[0194] Discussion

[0195] In most previous studies, quantitative metrics of muscle performance are generally limited to measurements of maximum displacement or maximum force in response to stimulated contraction. This is likely because the predominant method of quantitatively measuring engineered muscle contraction, beam bending, has key limitations that reduce the accuracy and reproducibility of dynamic tissue performance characterization. Specifically, beam-based methods are highly sensitive to the placement of and contact area between muscles and beams. Moreover, these methods rely on assumptions of linear elasticity and neglect the viscoelastic response of the polymers typically used to fabricate beams, reducing measurement accuracy at high frequency.

[0196] In this Example, principles of mechanical design have been leveraged to manufacture a linear elastic spring, in the form of a flexure, that can be coupled to modular muscle tissues via rigid posts. The design of the compliant mechanism determines their mechanical properties and can be precision machined in a cost-effective manner. Tuning the stiffness of the compliant mechanism to be much lower than muscle stiffness (e.g., -100X lower) enabled maximization of muscle twitch stroke by constraining the tissue’s degrees of freedom of motion without severely limiting contractile strain. As a result, ~5X higher contractile strain was observed on compliant mechanisms than previously reported using engineered muscle tissues fabricated from the mouse C2C12 line, outperforming even the previous theoretical maximum of untethered muscles. Strain measurement reproducibility was also significantly increased on compliant mechanisms as compared to untethered tissues or tissues tethered to beams, enabling precise diagnosis of differences between groups of tissues fabricated using slightly different biological reagents. Interestingly, maximizing the displacement of muscles per twitch also enabled decoding the dynamics of contraction across a range of frequencies. This Example reported key performance metrics (response time, contraction time, time at peak displacement / force, and relaxation time) that are necessary for predictive design of machines that leverage muscle tissues to produce a functional output. While past studies of engineered muscle suggested that time at peak force could only be increased by increasing the frequency of an input stimulus (to 10+ Hz), the results of this Example shows for the first time that modulating stimulus duty cycle can also control time at peak force with high precision, showcasing this in a demonstration of tetanic twitch induction at only 4 Hz. Thus, while twitch amplitude and time at peak force were always considered to be inextricably linked in the field of muscle biology, the results of this Example that show these two variables can be controlled independently by modulating stimulus duty cycle as well as frequency in a non-physiological manner.

[0197] Diving deeper into the frequency-dependent response of muscle, this Example also compares force, work, and power output at 1 and 4 Hz. The results show that while reducing the dynamic range of motion per twitch at higher frequency reduces work output per cycle, the power output of tissues is maximized at higher frequencies, corroborating previous observations of frequency-tunable performance in muscle-powered machines. It is important to note that a larger variability in force output per cycle was observed for 4 Hz stimulation (FIG. 5F), which can be attributed to the large variability in muscle relaxation dynamics (FIG. 4B). These results indicate that, for applications that require highly reproducible force output across many cycles, a lower frequency input stimulus may be more desirable.

[0198] Performance reproducibility across cycles is, however, not just dependent on input stimulus frequency, but also on the total duration of that stimulus. The compliant mechanism apparatus enabled stable longitudinal monitoring of muscle tissues over extended time periods, motivating the first investigation of the fatigue response of engineered muscle actuators. At 1 Hz, muscle displacement did not change significantly over 30 minutes of continuous stimulation, though plotting force-velocity curves over multiple cycles indicated performance variability increased over time. By contrast, at 4 Hz, muscle displacement reduced significantly after just 15 minutes of continuous stimulation and dropped further over the full 30-minute observation period. Fatigue in natural muscle is typically attributed to reduced availability of environmental glucose or fatigue of upstream motor neurons which trigger contraction. Since the latter is not a factor in this engineered system, one possible explanation for the observed fatigue is the limited rate of glucose diffusion from the surrounding media into the body of the mm-scale tissue, which may be observed sooner at 4 Hz stimulation since it elicits 4X the contractions of 1 Hz stimulation over the same time period. Alternatively, or additionally, fatigue in this system could be a consequence of the distribution of muscle fiber types within the engineered tissues. As muscles derived from the C2C12 line have been known to contain both slow-twitch and fasttwitch fibers, which are more and less fatigue resilient respectively, the exact distribution of fiber types within the muscles used in this Example could also play a role in regulating tissue-wide fatigue response to chronic stimulation.

[0199] This Example presents a simple and cost-efficient alternative to the gold standard method for measuring engineered tissue forces, beam bending, by showcasing how compliant mechanisms can be designed and robustly deployed for improving both the accuracy and precision of measuring muscle twitch magnitude and dynamics. This platform enables enhancing the contractile strain of muscle and decoding frequency-dependent and -independent acute and chronic twitch response. While this Example focuses on skeletal muscle, the system can be readily leveraged to study twitch forces generated by other types of contractile tissues, such as cardiac muscle, or even the forces generated by non-contractile tissues, such as the tensions generated by fibroblasts during wound healing. Finally, this Example demonstrates that compliant mechanisms can be used to precisely decode and significantly enhance the performance of contractile tissues, inspiring next-generation machines that couple compliant mechanisms with muscle actuators to address real- world challenges.

[0200] Experimental Methods

[0201] Fabrication of Compliant Mechanism Device The device shown in FIG. 2A was constructed from low-density polyethylene (flexure and base) and Teflon (pins). The modulus of elasticity for the acquired LDPE was 172 Mpa. The components were fabricated via precision CNC milling that is capable of meeting a tolerance of + / - 10 microns. The conjugated beams were machined to a thickness of 0.5mm and beam lengths along the conjugated chain varied between 25mm and 40mm. All beams were machined to a height of 3.35mm. To prevent the beams from deflecting during machining, the low-density polyethylene was fixated in place via double sided tape. A light finish pass was conducted on all beam side edges to prevent significant ‘spring’ due to deformation of the beams under cutting forces. Residual tape was removed via application of alcohol. Following fabrication, the device was adhered to the bottom of a 100mm Petri dish using polydimethylsiloxane (PDMS) (SYLGARD 184 silicone elastomer kit, Dow) with a 1:10 crosslinker:monomer ratio, cured at 37°C for 24 hours. The device was sterilized by submerging in 70% ethanol for 1 hour followed by UV light exposure for 1 hour.

[0202] Fabrication of Muscle Molds and Beams

[0203] Polylactic acid (PLA) templates for ring mold and beams were generated using computer- aided design (CAD) and 3D printed. Template dimensions were 10% larger than targeted dimensions to account for shrinkage of the PLA upon cooling. PDMS was cast inside the PLA templates at a crosslinkenmonomer ratio of 1:10 for ring molds and 1:33 for the beams. PDMS was also degassed before and after casting until bubbles were fully removed. PDMS-filled ring mold templates were cured at 37 °C for 24 hours, while PDMS beams were cured at 37 °C for 48 hours. After curing, the PDMS molds and beams were removed from the PLA casts manually using tweezers and sterilized by submerging in 70% ethanol for 1 hour, UV light exposure for 1 hour, and heat sterilization at 180°C for 40 minutes. Once sterile, the molds and beams were stored in lx DPBS (Gibco) at room temperature until use.

[0204] Fabrication of 3D Skeletal Muscle Rings Skeletal muscle rings were fabricated following a previously established protocol.

[0205] Optogcnctic C2C12 myoblasts expressing a mutant variant of Channclrhodopsin (ChR2[H134R]) were cultured in growth medium composed of DMEM with additions of 4.5g / L of glucose, L-glutamine, and sodium pyruvate (Coming) supplemented with 10% (v / v) fetal bovine serum (Sigma Aldrich), 1% (v / v) penicillin / streptomycin (Thermo Fisher Scientific), and 1% (v / v) L-glutamine (Thermo Fisher Scientific). Upon reaching 70% confluency, the cells were ready for muscle ring fabrication. First, thrombin (Sigma Aldrich) stock solution (lOOU / mL in 0.1% wt / v bovine serum albumin (Sigma Aldrich)), Matrigel (Coming), and 8mg of fibrinogen (Sigma Aldrich) were thawed and kept on ice. Depending on the sample, Growth Factor Reduced or High Concentration variants of Matrigel were used. The cells were released from their flasks with TypLE Express (Gibco) and portioned into 3xl06cell aliquots. These aliquots were centrifuged at lOOOrpm for 5min at room temperature to collect the cells as a pellet. The supernatants of these aliquots were aspirated, and the cells were resuspended in 59 pl of supplemented growth media containing 1 mg / mL 6-aminocaproic acid (Sigma Aldrich). At this point, the chilled fibrinogen was dissolved in ImL of supplemented growth media and vortexed thoroughly. Once the cells were resuspended, IpL of the thrombin stock solution, 90pL of Matrigel, and 150pL of the fresh fibrinogen stock solution were added to the cell suspension. The final volume of cell-gel solution (300pL) was dispensed into two PDMS ring molds. The filled molds were then incubated at 37°C for Ih and then submerged in supplemented growth medium (4mL / mold) to maintain cell viability. After setting for 24 hours in the ring mold, the matrix compacts into solid tissue. After an additional 24 hours, the muscles were changed to differentiation medium containing DMEM with 4.5g / L of glucose, L-glutamine, and sodium pyruvate, supplemented with 10% (v / v) horse serum (Thermo Fisher Scientific), 1% (v / v) penicillin / streptomycin, 1% (v / v) L-glutamine, 1 mg / mL of aminocaproic acid and 50 ng / mL of IGF-1 (Sigma Aldrich).

[0206] Optical Simulation and Video Recording of Muscle Muscles were manually transferred to beams or flexures and optically stimulated using a collimated 470-nm fiber optic LED (Thorlabs) placed 1.27cm above the surface of the medium containing the muscle. The stimulation platform was operated at frequencies of 1 or 4 Hz and a 20, 50, or 80% duty cycle for 0-30 consecutive minutes. Videos of muscle contraction were recorded using a stereomicroscope (Zeiss). Dim lighting on the stereomicroscope’s sample light was used as to limit exposure of the muscle to ambient light.

[0207] Muscle Fixing and Immunohistochemical Staining

[0208] Muscles were fixed in 4% paraformaldehyde for 24 hours, followed by preservation in 70% ethanol for another 24 hours on shaker tables. Samples were then transferred to cassettes, submerged in 70% ethanol, embedded in paraffin, and sliced into sections of 5pm thickness. The samples were then stained with DAPI to visualize nuclei and 1:200 MF-20 (Anti-Myosin 4, eBioscience, Invitrogen) to visualize mature muscle fibers.

[0209] MATLAB Automated Tracking of Contractile Displacement

[0210] To determine the magnitude of muscle ring contraction, the displacement of the PDMS beams and the device’s pillars were tracked frame-by-frame. An automated motion tracking MATLAB script was utilized to analyze the video recordings and quantitatively measure the displacement of the beam and device pillars during muscle contraction in two dimensions. The script allowed the selection of a trackable rectangular region within the video and calculated the pixel movement of the region, which is then converted to micrometers based on the scale of the video.

[0211] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the present disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

[0212] Embodiments: The following numbered embodiments fall within the scope of the invention:

[0213] 1. A device arranged to produce stiffness characteristics that regulate spatial displacement of a tissue in one or more directions, comprising: a solid support comprised of at least two rigid attachment points for supporting a tissue, wherein a first rigid attachment point positioned at one end of the solid support is connected to a compliant mechanism to allow displacement of a tissue and relative to a second rigid attachment point and in a direction x, wherein tissue displacement is constrained in at least one direction other than x, and optionally wherein the compliant mechanism has a stiffness value in the one or more directions that is lower than a stiffness value of the tissue in the one or more directions and at least one of the rigid attachment points has a stiffness value in the one or more directions that is higher than a stiffness value of the tissue in the one or more directions.

[0214] 2. A device, comprising:

[0215] (i) a first attachment point;

[0216] (ii) a second attachment point adjacent to the first attachment point, such that a tissue can interact with the first attachment point and the second attachment point; and

[0217] (iii) a compliant mechanism arranged to produce stiffness characteristics having a first end and a second end; wherein the first end of the compliant mechanism is connected to a solid support and the second end of the compliant mechanism is connected to the second attachment point; and wherein the compliant mechanism has a lower degree of stiffness in one direction than at least one other direction.

[0218] 3. The device of any one of embodiments 1-2, wherein the solid support is a base and wherein the base is attached to the first attachment point and the compliant mechanism, wherein the first attachment point is attached to the base by an adjustment mechanism. 4. The device of embodiment 3, wherein the adjustment mechanism is in a fixed position.

[0219] 5. The device of embodiment 3, wherein the adjustment mechanism is an adjustable cam, a wedge, a screw, or a lever.

[0220] 6. The device of embodiment 3 or embodiment 5, wherein the adjustment mechanism enables the first attachment point to be adjusted in at least one direction or orientation relative to the second attachment point.

[0221] 7. The device of embodiment 3 or embodiment 5, where the adjustment mechanism enables the distance between the first attachment point and the second attachment point to be adjusted to match the passive length of the tissue.

[0222] 8. The device of any one of embodiments 1-7, wherein the first attachment point is a rigid pillar.

[0223] 9. The device of any one of embodiments 1-8, wherein the second attachment point is a rigid pillar.

[0224] 10. The device of any one of embodiments 1-9, wherein the compliant mechanism has a rectilinear topology.

[0225] 11. The device of embodiment 10, wherein the rectilinear topology comprises parallel beams conjugated in series.

[0226] 12. The device of embodiment 11 or embodiment 12, wherein the rectilinear topology yields a low stiffness in one or more directions.

[0227] 13. The device of any one of embodiments 10-12, wherein the rectilinear topology yields a high stiffness in one or more directions.

[0228] 14. The device of any one of embodiments 10-13, wherein the rectilinear topology yields a low stiffness in one direction and a high stiffness in two directions.

[0229] 15. The device of any one of embodiments 1-9, wherein the compliant mechanism has a non-rectilinear topology.

[0230] 16. The device of any one of embodiments 1-15, wherein the compliant mechanism is made from a linear elastic material. 17. The device of embodiment 16, wherein the linear elastic material is a metal, a ceramic, or a biocompatiblc polymer.

[0231] 18. The device of any one of embodiments 1-17, wherein the compliant mechanism has a stiffness value in the one or more directions that is less than the stiffness value of the tissue in the one or more directions.

[0232] 19. The device of any one of embodiments 1-17, wherein the compliant mechanism has a stiffness value in the one or more directions that equals the stiffness value of the tissue in the one or more directions.

[0233] 20. The device of any one of embodiments 1-19, wherein the compliant mechanism has a stiffness value in the one or more directions that exceeds the stiffness value of the tissue in the one or more directions.

[0234] 21. The device of any one of embodiments 1-19, wherein the first and second rigid attachment points have a stiffness value in the one or more directions that is higher than a stiffness value of the tissue in the one or more directions.

[0235] 22. The device of any one of embodiments 16-21, wherein the linear elastic material is a low-density polyethylene.

[0236] 23. The device of any one of embodiments 2-22, wherein the first end of the compliant mechanism is connected to the base by a mount.

[0237] 24. The device of any one of embodiments 1-23, further comprising 2-4 additional attachment points capable of interacting with the tissue.

[0238] 25. The device of any one of embodiments 2-22, wherein the compliant mechanism has a lower degree of stiffness in one direction than in all other directions.

[0239] 26. The device of embodiment 1, wherein the tissue displacement is constrained all directions other than x.

[0240] 27. The device of any one of embodiments 1-26, wherein the device is monolithic.

[0241] 28. The device of any one of embodiments 1-26, wherein the device is modular.

[0242] 29. The device of any one of embodiments 1-28, wherein the device is configured to measure displacement, speed and / or acceleration. 30. The device of any one of embodiments 1-29, further comprising an actuator, wherein the actuator is configured to regulate spatial displacement of one or more components of the device.

[0243] 31. The device of any one of embodiments 1-30, wherein the stiffness of the compliant mechanism in the one or more directions relative to the stiffness of the tissue in the one or more directions is between 0.01 and 100.

[0244] 32. A method of assessing a tissue with a device, comprising:

[0245] (i) contacting a first attachment point and a second attachment point of a device with a tissue;

[0246] (ii) stimulating the tissue or device to produce a force one or more times; and,

[0247] (iii) measuring a property associated with displacement of the second attachment point relative to the first attachment point each of the one or more times, wherein the first attachment point and the second attachment point are rigid attachment points; wherein the second attachment point is connected to a base of the device by a compliant mechanism; and wherein the first attachment point is connected to the base of the device and wherein at least one of the rigid attachment points has a stiffness value in the direction of displacement that is greater than or equal to a stiffness value of the tissue in the direction of displacement.

[0248] 33. The method of embodiment 32, wherein the device is a device of any one of embodiments 1-31.

[0249] 34. The method of embodiment 35, wherein the first attachment point and the second attachment point are spaced at a fixed distance.

[0250] 35. The method of embodiment 34, wherein the fixed distance matches the passive length of the tissue.

[0251] 36. The method of embodiment 34, wherein the fixed distance is greater than the passive length of the tissue.

[0252] 37. The method of embodiment 32, wherein the first attachment point is connected to the compliant mechanism by an adjustment mechanism. 38. The method of embodiment 37, wherein the adjustment mechanism is a cam, a wedge, a screw, or a lever.

[0253] 39. The method of embodiment 37 or embodiment 38, wherein the adjustment mechanism enables the first attachment point to be adjusted in at least one direction or orientation relative to the second attachment point.

[0254] 40. The method of any one of embodiments 32-39, wherein the compliant mechanism has a rectilinear topology.

[0255] 41. The method of embodiment 40, wherein the rectilinear topology comprises parallel beams conjugated in series.

[0256] 42. The method of any one of embodiments 32-39, wherein the compliant mechanism has a non-rectilinear topology.

[0257] 43. The method of any one of embodiments 32-42, wherein the compliant mechanism has a stiffness in the direction of displacement that is less than the stiffness of the tissue in the direction of displacement.

[0258] 44. The method of embodiment 43, wherein when the compliant mechanism has a stiffness in the direction of displacement that is less than the stiffness of the tissue in the direction of displacement, displacement is maximized.

[0259] 45. The method of any one of embodiments 32-42, wherein the compliant mechanism has a stiffness in the direction of displacement that equals the stiffness of the tissue in the direction of displacement.

[0260] 46. The method of embodiment 45, wherein when the compliant mechanism has a stiffness in the direction of displacement that equals the stiffness of the tissue in the direction of displacement work output is maximized.

[0261] 47. The method of any one of embodiments 32-42, wherein the compliant mechanism has a stiffness in the direction of displacement that exceeds the stiffness of the tissue in the direction of displacement. 48. The method of embodiment 47, wherein when the compliant mechanism has a stiffness in the direction of displacement that exceeds the stiffness of the tissue in the direction of displacement, force output is maximized.

[0262] 49. The method of any one of embodiments 32-48, wherein the tissue is attached to the first attachment point and the second attachment point.

[0263] 50. The method of any one of embodiments 32-48, wherein the tissue is placed around the first attachment point and the second attachment point.

[0264] 51. The method of any one of embodiments 32-50, wherein the tissue is a contractile tissue.

[0265] 52. The method of embodiment 51, where the contractile tissue is a skeletal muscle tissue, cardiac muscle tissue, or smooth muscle tissue.

[0266] 53. The method of any one of embodiments 32-50, wherein the tissue sample is a non-contractile tissue.

[0267] 54. The method of any one of embodiments 32-50, wherein the tissue is a connective tissue, an epithelial tissue, or a tissue of the nervous system.

[0268] 55. The method of embodiment 54, wherein the connective tissue is a tendon, a ligament, a fibroblast, or cartilage.

[0269] 56. The method of embodiment 54, wherein the epithelial tissue is skin or blood vessels.

[0270] 57. The method of embodiment 54, wherein the tissue of the nervous system is a motor neuron, glia, or ganglion.

[0271] 58. The method of any one of embodiments 32-57, wherein the force is a normal force or a shear force.

[0272] 59. The method of any one of embodiments 32-58, wherein the tissue or device is stimulated optically, chemically, electrically, mechanically, thermally, or magnetically.

[0273] 60. The method of any one of embodiments 32-58, where the tissue or device is stimulated with fiber optics. 61 . The method of any one of embodiments 32-60, wherein the force is produced by the compliant mechanism.

[0274] 62. The method of any one of embodiments 32-61, wherein the force is produced by the tissue.

[0275] 63. The method of any one of embodiments 32-62, wherein displacement can be measured using image tracking.

[0276] 64. The method of any one of embodiments 32-63, wherein the force can be produced by low-frequency stimulation.

[0277] 65. The method of embodiment 64, wherein the low-frequency stimulation is less than or equal to 1 Hz.

[0278] 66. The method of any one of embodiments 32-63, wherein the force can be produced by high-frequency stimulation.

[0279] 67. The method of embodiment 66, wherein the high-frequency stimulation is greater than or equal to 2 Hz.

[0280] 68. The method of any one of embodiments 32-67, wherein the property associated with displacement of the second attachment point relative to the first attachment point is measured as displacement, speed, velocity, acceleration, or combinations thereof.

[0281] 69. A method for evaluating effect of a biologically active substance on a tissue, comprising:

[0282] (i) contacting a tissue with a biologically active substance; wherein the tissue is positioned on a device and in contact with at least two attachment points on the device, and wherein a compliant mechanism is connected to at least one of the two attachment points on the device, wherein at least one of the first attachment point and the second attachment point have a stiffness in a direction of desired motion that is greater than the stiffness of the tissue in the direction of desired motion;

[0283] (ii) producing a force one or more times; and

[0284] (iii) measuring a property associated with displacement of the second attachment point relative to the first attachment point each of the one or more times, wherein the property associated with displacement provides information on the effect of the biologically active substance on the tissue.

[0285] 70. The method of embodiment 69, wherein the device is a device of any one of embodiments 1-31.

[0286] 71. The method of embodiment 69 or embodiment 79, wherein the biologically active substance is a small molecule, a nucleic acid, or a protein.

[0287] 72. The method of embodiment 71, wherein the nucleic acid is a gene therapy product.

[0288] 73. The method of any one of embodiments 69-71, wherein the tissue is attached to the first attachment point and the second attachment point.

[0289] 74. The method of any one of embodiments 69-71, wherein the tissue is placed around the first attachment point and the second attachment point.

[0290] 75. The method of any one of embodiments 69-74, wherein the tissue is a contractile tissue.

[0291] 76. The method of embodiment 75, where the contractile tissue is a skeletal muscle tissue, cardiac muscle tissue, or smooth muscle tissue.

[0292] 77. The method of any one of embodiments 69-74, wherein the tissue sample is a non-contractile tissue.

[0293] 78. The method of any one of embodiments 69-74, wherein the tissue is a connective tissue, an epithelial tissue, or a tissue of the nervous system.

[0294] 79. The method of embodiment 78, wherein the connective tissue is a tendon, a ligament, a fibroblast, or cartilage.

[0295] 80. The method of embodiment 78, wherein the epithelial tissue is skin or blood vessels.

[0296] 81. The method of embodiment 78, wherein the tissue of the nervous system is a motor neuron, glia, or ganglion.

[0297] 82. The method of any one of embodiments 69-81, wherein the force is a normal force or a shear force. 83. The method of any one of embodiments 69-82, wherein the force is stimulated optically, chemically, electrically, mechanically, thermally, or magnetically.

[0298] 84. The method of any one of embodiments 69-82, where the force is stimulated with fiber optics.

[0299] 85. The method of any one of embodiments 69-84, wherein the force is produced by the compliant mechanism.

[0300] 86. The method of any one of embodiments 69-85, wherein the force is produced by the tissue.

[0301] 87. The method of any one of embodiments 69-86, wherein displacement can be measured using image tracking.

[0302] 88. The method of any one of embodiments 69-87, wherein the force can be produced by low-frequency stimulation.

[0303] 89. The method of embodiment 88, wherein the low-frequency stimulation is less than or equal to 1 Hz.

[0304] 90. The method of any one of embodiments 69-87, wherein the force can be produced by high-frequency stimulation.

[0305] 91. The method of embodiment 90, wherein the high-frequency stimulation is greater than or equal to 2 Hz.

[0306] 92. The method of any one of embodiments 69-91, wherein the property associated with displacement of the second attachment point relative to the first attachment point is measured as displacement, speed, velocity, acceleration, or combinations thereof.

[0307] EQUIVALENTS AND SCOPE

[0308] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims. In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group arc considered satisfied if one, more than one, or all of the group members arc present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0309] Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the present disclosure, or aspects of the present disclosure, is / are referred to as comprising particular elements and / or features, certain embodiments of the present disclosure or aspects of the present disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the terms “comprising” and “containing” are intended to be open and permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0310] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the present disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.

[0311] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

CLAIMS1. A device arranged to produce stiffness characteristics that regulate spatial displacement of a tissue in one or more directions, comprising: a solid support comprised of at least two rigid attachment points for supporting a tissue, wherein a first rigid attachment point positioned at one end of the solid support is connected to a compliant mechanism to allow displacement of a tissue and relative to a second rigid attachment point and in a direction x, wherein tissue displacement is constrained in at least one direction other than x, and optionally wherein the compliant mechanism has a stiffness value in the one or more directions that is lower than a stiffness value of the tissue in the one or more directions and at least one of the rigid attachment points has a stiffness value in the one or more directions that is higher than a stiffness value of the tissue in the one or more directions.

2. A device, comprising:(i) a first attachment point;(ii) a second attachment point adjacent to the first attachment point, such that a tissue can interact with the first attachment point and the second attachment point; and(iii) a compliant mechanism arranged to produce stiffness characteristics having a first end and a second end; wherein the first end of the compliant mechanism is connected to a solid support and the second end of the compliant mechanism is connected to the second attachment point; and wherein the compliant mechanism has a lower degree of stiffness in one direction than at least one other direction.

3. The device of any one of claims 1-2, wherein the solid support is a base and wherein the base is attached to the first attachment point and the compliant mechanism, wherein the first attachment point is attached to the base by an adjustment mechanism, optionally wherein the adjustment mechanism is: a) in a fixed position, b) an adjustable cam, a wedge, a screw, or a lever,c) wherein the adjustment mechanism enables the first attachment point to be adjusted in at least one direction or orientation relative to the second attachment point, or d) enables the distance between the first attachment point and the second attachment point to be adjusted to match the passive length of the tissue.

4. The device of any one of claims 1-3, wherein the first attachment point is a rigid pillar and / or wherein the second attachment point is a rigid pillar.

5. The device of any one of claims 1-4, wherein the compliant mechanism has a rectilinear topology, wherein the rectilinear topology comprises parallel beams conjugated in series, wherein the rectilinear topology yields: a) a low stiffness in one or more directions b) a high stiffness in one or more directions, or c) a low stiffness in one direction and a high stiffness in two directions.

6. The device of any one of claims 1-4, wherein the compliant mechanism has a non-rectilinear topology.

7. The device of any one of claims 1-6, wherein the compliant mechanism is made from a linear elastic material and optionally wherein the linear elastic material is a metal, a ceramic, or a biocompatible polymer.

8. The device of any one of claims 1-7, wherein the compliant mechanism has a stiffness value in the one or more directions: a) that is less than the stiffness value of the tissue in the one or more directions, b) that equals the stiffness value of the tissue in the one or more directions, c) that exceeds the stiffness value of the tissue in the one or more directions, or d) that is higher than a stiffness value of the tissue in the one or more directions.

9. The device of any one of claims 7-8, wherein the linear clastic material is a low- density polyethylene.

10. The device of any one of claims 2-9, wherein the first end of the compliant mechanism is connected to the base by a mount.

11. The device of any one of claims 1-10, further comprising 2-4 additional attachment points capable of interacting with the tissue.

12. The device of any one of claims 2-11, wherein the compliant mechanism has a lower degree of stiffness in one direction than in all other directions or wherein the tissue displacement is constrained all directions other than x.

13. The device of any one of claims 1-12, wherein the device is monolithic or modular.

14. The device of any one of claims 1-13, wherein the device is configured to measure displacement, speed and / or acceleration.

15. The device of any one of claims 1-14, further comprising an actuator, wherein the actuator is configured to regulate spatial displacement of one or more components of the device.

16. The device of any one of claims 1-15, wherein the stiffness of the compliant mechanism in the one or more directions relative to the stiffness of the tissue in the one or more directions is between 0.01 and 100.

17. The device of any one of claims 1- 16, wherein the first and second attachment points arc sized to fit within a well of a multiwcll plate, such as a 24 or 96 well plate.

18. The device of claim 16 or 17, wherein the device is sized to function with a single well of a multiwell plate, such as a 24 or 96- well plate.

19. A method of assessing a tissue with a device, comprising:(i) contacting a first attachment point and a second attachment point of a device with a tissue;(ii) stimulating the tissue or device to produce a force one or more times; and,(iii) measuring a property associated with displacement of the second attachment point relative to the first attachment point each of the one or more times, wherein the first attachment point and the second attachment point are rigid attachment points; wherein the second attachment point is connected to a base of the device by a compliant mechanism; and wherein the first attachment point is connected to the base of the device and wherein at least one of the rigid attachment points has a stiffness value in the direction of displacement that is greater than or equal to a stiffness value of the tissue in the direction of displacement.

20. The method of claim 19, wherein the device is a device of any one of claims 1-18.

21. The method of claim 20, wherein the first attachment point and the second attachment point are spaced at a fixed distance and wherein the fixed distance matches the passive length of the tissue or wherein the fixed distance is greater than the passive length of the tissue.

22. The method of any one of claims 19-21 , wherein the tissue is a contractile tissue, optionally wherein the contractile tissue is a skeletal muscle tissue, cardiac muscle tissue, or smooth muscle tissue.

23. The method of any one of claims 19-21, wherein the tissue sample is a non- contractile tissue, optionally wherein the tissue is a connective tissue, an epithelial tissue, or a tissue of the nervous system, optionally wherein the connective tissue is a tendon, a ligament, a fibroblast, or cartilage, wherein the epithelial tissue is skin or blood vessels, and wherein the tissue of the nervous system is a motor neuron, glia, or ganglion.24 The method of any one of claims 19-23, wherein the force is a normal force or a shear force.

25. The method of any one of claims 19-24, wherein the tissue or device is stimulated optically, such as stimulation with fiber optics, chemically, electrically, mechanically, thermally, or magnetically.

26. The method of any one of claims 19-25, wherein the force is produced by the compliant mechanism or by the tissue.

27. The method of any one of claims 19-26, wherein displacement can be measured using image tracking.

28. The method of any one of claims 19-27, wherein the force can be produced by low-frequency stimulation optionally, wherein the low-frequency stimulation is less than or equal to 1 Hz.

29. The method of any one of claims 19-27, wherein the force can be produced by high-frequency stimulation optionally wherein the high-frequency stimulation is greater than or equal to 2 Hz.

30. The method of any one of claims 19-27, wherein the property associated with displacement of the second attachment point relative to the first attachment point is measured as displacement, speed, velocity, acceleration, or combinations thereof.

31. A method for evaluating effect of a biologically active substance on a tissue, comprising:(i) contacting a tissue with a biologically active substance; wherein the tissue is positioned on a device and in contact with at least two attachment points on the device, and wherein a compliant mechanism is connected to at least one of the two attachment points on the device, wherein at least one of the first attachment point and the second attachment point have a stiffness in a direction of desired motion that is greater than the stiffness of the tissue in the direction of desired motion;(ii) producing a force one or more times; and(iii) measuring a property associated with displacement of the second attachment point relative to the first attachment point each of the one or more times, wherein the property associated with displacement provides information on the effect of the biologically active substance on the tissue optionally wherein the biologically active substance is a small molecule, a nucleic acid, a gene therapy product, or a protein.

32. The method of claim 31, wherein the device is a device of any one of claims 1-18.

33. A device, comprising:(i) a solid support comprising a first attachment point and a second attachment point adjacent to the first attachment point, such that a tissue can interact with the first attachment point and the second attachment point; and(ii) a compliant mechanism connected to the second attachment point and structurally between the first and second attachment point so that it takes significant load from that applied by the tissue, and(iii) a sensing mechanism comprised of a strain gauge that is placed at a location within a structural loop between the first attachment point and the second attachment point.

34. The device of claim 33, wherein the device comprises 1-4 strain gauges.

35. The device of any one of claims 33-34, wherein the sensing mechanism is a comprised of a straight beam with four semiconductor strain gauges.

36. The device of any one of claims 33-35, wherein the strain gauges are arranged as a bridge circuit, with two of the four semiconductor strain gauges arranged on each end on opposite sides.

37. The device of any one of claims 33-36, wherein the device comprises a low-density polyethylene material.

38. The device of any one of claims 33-37 wherein the compliant mechanism has a first end and a second end and is arranged to produce stiffness characteristics; wherein the first end of the compliant mechanism is connected to a solid support and the second end of the compliant mechanism is connected to the second attachment point; and wherein the compliant mechanism has a lower degree of stiffness in a direction x that is nominally aligned with the load and displacement of the tissue.

39. The device of any one of claims 33-37, wherein the first attachment point allows displacement of the tissue relative to a second attachment point and in a direction x, wherein the relative displacement of the tissue ends is constrained in at least one direction other than x, and optionally wherein the compliant mechanism has a stiffness value in the one or more directions that is lower than a stiffness value of the tissue in the one or more directions, and optionally wherein the relative displacement of the tissue ends is constrained in all other directions other than x.

40. The device of any one of claims 33-39 wherein the strain gauge comprises D-type mono-crystalline silicon.

41. The device of any one of claims 33-39, wherein the strain gauge comprises metallic strain gauges.

42. The device of any one of claims 33-40, wherein the strain gauge has welded gold alloy leads.

43. The device of any one of claims 33-42, wherein the structural loop carries a load placed upon it, that is the parts of the structure that feel the force exerted by the muscle44. The device of any one of claims 33-43, further comprising a tensioning mechanism, wherein the tensioning mechanism is arranged to connect to at least one of the attachment points.

45. The device of claim 44, further comprising a variable stiffness mechanism, wherein the tensioning mechanism and the variable stiffness mechanism each are arranged to connect to one of the at least two attachment points for supporting a tissue.

46. A device for regulating spatial displacement of a tissue in one or more directions, comprising a base having a circular geometry with an outer perimeter and a grounded central point, a tensioning mechanism and a variable stiffness mechanism, wherein the tensioning mechanism and the variable stiffness mechanism each connect to one of at least two attachment points for supporting a tissue.

47. The device of claim 46, wherein the device comprises a first attachment point for supporting a tissue, attached at a first position on the outer perimeter of the base and a second attachment point for supporting the tissue, attached at a second position on the outer perimeter of the base and arranged relative to the first attachment point, such that the tissue can interact with the first attachment point and the second attachment point.

48. The device of any one of claims 46-47, wherein the tensioning mechanism comprises a tension arm connected to the grounded central point by a rotary tension flexure bearing (TFB).

49. The device of any one of claims 46-48, wherein the variable stiffness mechanism comprises a variable stiffness arm and a variable stiffness flexure stage (VSFS), wherein the variable stiffness arm is connected to the grounded central point and also contacts a variable stiffness actuator point (VSAP).

50. The device of claim 49, wherein the variable stiffness arm varies in cross-section along its length.

51. The device of any one of claims 49-50, wherein the VSFS and the variable stiffness arm are arranged relative to one another such that a radial gap exists between them.

52. The device of any one of claims 49-51 , wherein an actuator interfaces with the VSFS at a variable stiffness actuator point (VSAP).

53. The device of claim 48, wherein an actuator interfaces with the tensioning arm at a tensioning actuation point (TAP).

54. The device of any one of claims 46-53, further comprising an actuator, wherein the actuator has one or two degrees of freedom.

55. The device of claim 54, wherein the actuator comprises a motor housing and two shafts, wherein one of the shafts is connected to the actuation point for the TAP and the other of the shafts is connected to the actuation point for the VSAP.

56. The device of claim 55, wherein the two shafts are arranged to rotate independent of one another.

57. A device, comprising:(i) a multi- well plate lid,(ii) a first attachment point and a second attachment point adjacent to the first attachment point, such that a tissue can interact with the first attachment point and the second attachment point attached to a bottom surface of the multi-well plate lid or otherwise positioned at the bottom surface of the lid; and(iii) a compliant mechanism connected to the first attachment point.

58. A method of assessing a tissue with a device, comprising:(i) contacting a first attachment point and a second attachment point of a device with a tissue, wherein the second attachment point is connected to a compliant mechanism comprising more than one functional beam which collectively provide a stiffness value;(ii) stimulating the tissue or device to produce a force one or more times; and,(iii) measuring a property associated with displacement of the second attachment point relative to the first attachment point each of the one or more times, wherein the stiffness value of the compliant mechanism is modified and steps (ii) and (iii) are repeated.

59. The method of claim 58, wherein the stiffness value of the compliant mechanism is reduced by reducing the number of functional beams, physically disrupting the body of the beams or their structural connections.

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