Reversing aging with ultrasound exposure

Abstract

The present invention includes a non-invasive method of treating aging using low frequency structured and modulated ultrasound. Ultrasound exposure can cause mechanical stretching of cells even within the body of an organism, thereby reversing the characteristics of senescent cells and countering their effects on the function of the tissue in which they reside. Reversal of aging by ultrasound includes at least one of activating cell proliferation, reducing cell size, increasing secretion of growth factors, increasing mitochondrial fission, and / or promoting wound healing. Ultrasound treatment improves aging, specific organ function, wound healing, and allows for a more significant increase in normal cells in vitro. Ultrasound can be delivered to the whole organism, or smaller targeted ultrasound devices can be made for specific organ or cell applications.

Description

[Technical field]

[0001] The present invention relates generally to the field of aging, and in particular to reversing aging through ultrasound exposure.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT none

[0003] INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC none [Background technology]

[0004] Without limiting the scope of the invention, its background is described in connection with aging.

[0005] As people's life expectancy increases and the population ages, more and more people suffer from age-related disorders that impair their function and overall quality of life. Many of the problems associated with aging can be explained as a result of cellular senescence. Senescent cells do not grow or function properly and secrete factors that increase the senescence of neighboring cells.

[0006] What is needed are treatments for aging adults that will reduce the fraction of senescent cells in the body, thereby reducing the deleterious effects of aging and improving quality of life. There are many factors that can increase lifespan, but these have not been generally characterized for their effects on quality of life.

[0007] Currently, there are two main approaches to combat aging that appear to increase quality of life: exercise and senolysis. Continuous exercise in older adults has many benefits and helps to counter the effects of aging on physical and mental performance. Senolysis is a drug or small molecule that triggers apoptosis of senescent cells and has been shown to improve the performance of older mice by reducing the fraction of senescent cells in the body. Some people consider exercise to be senolysis, so the two approaches may act through a common target: reducing the fraction of senescent cells. In view of the weakness of these approaches, exercise is difficult to sustain due to injuries, lifestyle issues, and personal will. Unfortunately, senolysis is difficult to target only senescent cells and often has side effects.

[0008] Despite these advances, new treatment methodologies are needed that help reduce the rate of aging, reverse cellular senescence, and / or reduce or eliminate agents released by senescent cells that increase the rate of aging. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention uses ultrasound irradiation to mimic the mechanical effect of exercise on aging.It is demonstrated herein that the mechanical effect of ultrasound reverses cellular aging without killing cells.Another advantage of low-frequency ultrasound (US) is that it can penetrate the whole body and act on aging cells inside, reversing aging in situ and restoring cells to a functional state.

[0010] As embodied and broadly described herein, one aspect of the disclosure relates to a non-invasive method of treating aging, comprising mechanically stretching at least one viable cell in an amount sufficient to retard at least one characteristic of aging. In one aspect, the at least one characteristic of aging is selected from reduced cellular senescence, increased cell division, reduced cell size, increased secretion of growth factors, reduced secretion of senescence factors, reduced mitochondrial fusion, increased mitochondrial fission, or enhanced wound healing. In another aspect, mechanically stretching at least one viable cell is by applying repetitive low-frequency ultrasound waves configured to target an area requiring treatment for aging, the area being targeted by one or more ultrasound sources directed at the target from one or more directions. In another aspect, the immersion design for treatment is optimized for treating wounds and ulcers, and may include one or more chambers suitable for complete immersion, generally shallow, with absorber shielding across the treatment area, and the transducer and absorber may be in contact with the patient or insulated by the immersion medium. In another embodiment, the step of mechanically stretching the at least one viable cell is by applying repetitive low frequency ultrasound selected from at least one of a standing wave pattern, a shock wave, a rapid sound change, a square wave, a sawtooth wave, a random wave, or an undulating low-high intensity beat. In another embodiment, the step of mechanically stretching the at least one viable cell is repeated for a period of time selected from the group consisting of once a day, about once every two days, about once every three days, about once a week, and about twice a week by applying repetitive low frequency ultrasound with a duration between about 5 minutes and about 60 minutes. In another aspect, the step of mechanically stretching the at least one viable cell is by applying repetitive low frequency ultrasound for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90 minutes, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours.In another embodiment, the step of mechanically stretching the at least one viable cell is by applying repetitive low frequency ultrasound at 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900 kHz, 1 MHz, or more. In another embodiment, the step of mechanically stretching the at least one viable cell is by applying repetitive low frequency ultrasound at 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1,000, 5,000, 10,000, or <500 mW / cm. 2 In another embodiment, the at least one viable cell is a cell line or cell clone (or other primary cell) or is present in a tissue, an organ, a limb, or the whole body. In another embodiment, at least a portion of the step of mechanically stretching the at least one viable cell is by repetitive low frequency ultrasound treatment applied simultaneously with one or more active agents that reduce cellular senescence. In another embodiment, the active agent is a supernatant of cells treated with repetitive low frequency ultrasound treatment. In another embodiment, the step of mechanically stretching the at least one viable cell is by one or more ultrasound transducers configured to create a sequence of waves of programmed cycles; one or more ultrasonic transducers attached to the robotic arm; one or more ultrasonic transducers, the robotic arm being controlled to position the ultrasonic transducers in a desired position or orientation; One or more ultrasonic transducers, which may be phased array ultrasonic transducers or which are hermetically sealed and water resistant; a repetitive low frequency ultrasound treatment delivered from at least one of The frequency, magnitude, and duration of the periodic force are determined at least in part based on the type of target cell or based on the output of a feedback sensor near the treatment area; or The wavelength of the waves is on the order of the size of the organ, and the amplitude is on the order of a single cell. In another embodiment, the step of mechanically stretching at least one viable cell is a sequence of waves of programmed cycles created according to a type of treatment plan or therapeutic procedure, the treatment plan being created using one or more machine learning techniques, low frequency modulation of sound waves, periodic on-off patterns with set duty cycles, variations in amplitude, frequency, and phase; superposition of several carrier frequencies, each with variable phase and center frequency and relative intensity; modulation by an external input signal, modulation by an audible sound signal. In another embodiment, the method further includes the step of preparing a controller, the controller being configured to control one or more ultrasonic transducers based on sensor data, and being integrated with one or more ultrasonic generators, and including a remote driver that limits energy from one or more ultrasonic generators to provide patient safety; a neutral driver that always zeros the voltage between two or more ultrasonic transducer elements, or is connected to one or more transducer stacks that reduces the peak voltage used by one or more ultrasonic generators. In another aspect, the at least one viable cell is a plurality of viable cells, and the method further comprises applying repetitive low-frequency ultrasound treatment to the plurality of in vivo viable cells. In another aspect, the step of mechanically stretching the at least one viable cell is a repetitive low-frequency ultrasound treatment delivered to at least a localized region of the subject's body. In another aspect, the repetitive low-frequency ultrasound treatment is provided by a piezoelectric transducer, a voice coil, a capacitive membrane, fluid instability, hydraulics with shutters, a pneumatic device, a spark discharge, chemically created pressure waves, engine driving sounds, induced muscle tension, a transducer array, a phased array with a synthetic aperture, or a harmonic of frequency. In another aspect, the at least one viable cell is a plurality of viable cells, and the step of applying repetitive low-frequency ultrasound treatment extends the replicative life span of the at least one viable cell.In another embodiment, the at least one viable cell is a plurality of viable cells, and applying the repetitive low-frequency ultrasound treatment reverts the plurality of viable cells to a younger phenotype. In another embodiment, the method further comprises providing a mechanism that is a material suitable for at least one of absorbing or isolating the repetitive low-frequency ultrasound treatment to reduce or deflect the acoustic waves after treatment of the patient, attenuating the acoustic waves or directing the acoustic waves away from the treatment site, or includes one or more elements that convert residual acoustic energy into heat.

[0011] As embodied and broadly described herein, one aspect of the present disclosure relates to a method for reducing cellular senescence, comprising applying repetitive low-frequency ultrasound treatment to at least one viable cell, wherein the repetitive low-frequency ultrasound treatment retards at least one aging characteristic. In one aspect, the wavelength is equal to or greater than the average cell diameter. In another aspect, the at least one aging characteristic is selected from reducing cellular senescence, increasing cell division, reducing cell size, increasing secretion of growth factors, reducing secretion of senescence factors, preventing mitochondrial fusion, increasing mitochondrial division, or enhancing wound healing. In another aspect, the applying repetitive low-frequency ultrasound treatment is configured to target the area requiring treatment for aging, and the area is targeted by one or more ultrasound sources that are directed toward the target from one or more directions. In another aspect, the applying repetitive low-frequency ultrasound treatment is selected from at least one of a standing wave pattern, a shock wave, a rapid transition, a square wave, a sawtooth wave, a random wave, or an undulating low-high intensity beat. In another embodiment, the step of applying the repetitive low frequency ultrasound treatment lasts between about 5 minutes and about 30 minutes and is repeated for a period selected from the group consisting of once a day, once every 2 days, once every 3 days, once a week, and twice a week. In another embodiment, the step of applying the repetitive low frequency ultrasound treatment lasts for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90 minutes, or for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 24 hours. In another embodiment, the step of applying repetitive low frequency ultrasound treatment is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900 kHz, 1 MHz, or more. In another embodiment, the step of applying repetitive low frequency ultrasound treatment is 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1,000, 5,000, 10,000, or <500 mW / cm 2In another embodiment, the at least one viable cell is a cell line or cell clone (such as a primary cell) or is present in a tissue, an organ, a limb, or the whole body. In another embodiment, at least a portion of the application of the repetitive low-frequency ultrasound treatment is applied simultaneously with an active agent that reduces cellular senescence. In another embodiment, the active agent is a supernatant of cells treated with the repetitive low-frequency ultrasound treatment. In another embodiment, the repetitive low-frequency ultrasound treatment is applied until the at least one viable cell reaches cellular senescence to delay at least one aging characteristic. In another embodiment, the repetitive low-frequency ultrasound treatment is applied by one or more ultrasound transducers configured to create a sequence of waves of programmed cycles; one or more ultrasonic transducers attached to the robotic arm; one or more ultrasonic transducers, the robotic arm being controlled to position the one or more ultrasonic transducers in a desired location or orientation; One or more ultrasonic transducers that are either phased array ultrasonic transducers or are hermetically sealed and water resistant delivered from at least one of The frequency, magnitude, and duration of the periodic force are determined at least in part based on the type of target cell or based on the output of a feedback sensor at or near the treatment area; or The wavelength of the wave is on the order of the size of the organ, and the amplitude is on the order of a single cell. In another aspect, the repetitive low frequency ultrasound treatment is at least one of a sequence of waves of a programmed cycle created according to a type of treatment plan or therapeutic procedure; the treatment plan is created using one or more machine learning techniques, low frequency modulation of sound waves, periodic on-off patterns with a set duty cycle, variations in amplitude, frequency, and phase; superposition of several carrier frequencies, each with variable phase, center frequency, and relative intensity; a signal modulated by an external input signal, or a signal modulated by an audible sound. In another aspect, the method further includes the step of preparing a controller, the controller configured to control one or more ultrasound transducers based on the sensor data, integrated with one or more ultrasound generators, a remote driver that limits energy from one or more ultrasound generators to provide patient safety; a neutral driver that always zeros the voltage between one or more ultrasound transducer elements or is connected to one or more transducer stacks that reduces the peak voltage used by one or more ultrasound generators. In another aspect, the at least one viable cell is a plurality of viable cells, and the method further includes the step of applying the repetitive low frequency ultrasound treatment to the plurality of in vivo viable cells. In another aspect, the repetitive low-frequency ultrasound treatment is delivered to at least a localized region of the patient's body. In another aspect, the repetitive low-frequency ultrasound treatment is provided by a piezoelectric transducer, a voice coil, a capacitive membrane, fluid instability, hydraulics with shutters, pneumatics, spark discharge, chemically generated pressure waves, engine driving sounds, induced muscle tension, phased arrays, phased arrays with one or more synthetic apertures, or harmonics. In another aspect, the at least one viable cell is a plurality of viable cells, and applying the repetitive low-frequency ultrasound treatment extends the replicative lifespan of the at least one viable cell. In another aspect, the at least one viable cell is a plurality of viable cells, and applying the repetitive low-frequency ultrasound treatment reverts the plurality of viable cells to a younger phenotype.In another aspect, the method further comprises providing a mechanism that is a material suitable for at least one of absorbing or isolating repetitive low frequency ultrasonic treatment to reduce or deflect sound waves after treatment of the patient, attenuating sound waves or directing sound waves away from the treatment site, or includes one or more elements that convert residual sound energy into heat.In another aspect, the method further comprises providing an immersion vessel for treating wounds and ulcers that includes one or more chambers that are partially or completely immersed with an absorber shield around the treatment area, and the one or more transducers and the one or more absorbers may be in contact with the patient or are insulated by the immersion medium.

[0012] For a more complete understanding of the nature and advantages of the present invention, reference should be made to the following detailed description of the invention taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0013] [Figure 1] Figures 1A-1H show the characterization of senescent cells. (Figure 1A) Brightfield images of senescent cells induced by H2O2, sodium butyrate (SB), doxorubicin (Dox), and bleomycin sulfate (BS). Scale bar = 300 μm. (Figure 1B) Quantification of proliferation shows no senescent growth after 48 h of incubation. (Figure 1C) Senescent cells become larger compared to normal control cells. (Figure 1D) Quantification of average cell volume of cells treated with bleomycin sulfate compared to control cells. (Figure 1E) Conditioned medium from senescent cells inhibits the growth of normal proliferating cells. (Figure 1F) SA-b-galactosidase staining of control (proliferating) cells and senescent cells treated with BS. Scale bar = 300 μm. (Figure 1G) Levels of b-galactosidase senescence marker in SCs induced with H2O2 and SB. Scale bar = 300 μm. Data from (FIGS. 1B)-(1G) A minimum of 150 cells from n=2 experiments were analyzed for spreading area and cell volume. [Diagram 2]Figures 2A-2G show that ultrasound reverses cellular senescence. (Figure 2A) Schematic of the senescence reversal experiment. SB-treated Vero cells were treated with low-frequency ultrasound (LFU) and passaged every 48 h for 8-10 days. (Figure 2B) Graph showing cell proliferation as fold change over 48 h. Passage of treated cells from P0 to P3 every 48 h. (Figure 2C) Cell area (LFU) of LFU-treated senescent cells largely recovers to normal by P3. (Figure 2D) Representative anti-p21 immunofluorescence images of P3 control and LFU-treated cells. Scale bar = 300 μm. (Figure 2E) Quantification of fluorescence intensity of control and LFU-treated P3 cells stained with p21. Mean ± SD of >200 cells in each condition is shown. (Figure 2F) Ultrasound increased proliferation as determined by EDU staining. (Figure 2G) Quantification of EDU-positive P3 cells. All graphs are plotted as mean ± SD. A minimum of 200 cells were analyzed for each condition. [Diagram 3]Figures 3A-3H show that USS increases the growth of senescent cells while inhibiting the control senescence-associated secretory phenotype (SASP). (Figure 3A) Schematic of the experiment. SCs were cultured in growth medium for 24 h and then treated with LFU for 30 min. After LFU treatment, the supernatant was collected (S0) and again after 24 h (S24). To confirm the effect of LFU treatment, the supernatants S0 and S24 were used to confirm the growth of non-senescent control cells. (Figure 3B) Representative brightfield images of control cells after 48 h of incubation in normal growth medium, S0, and S24. (Figure 3C and 3D) Quantification of control cell numbers shows growth and normal size in control and S24 medium, but reduced growth and increased size in S0. (Figure 3E). Schematic timeline and strategy of LFU treatment of normal proliferating cells. Control cells were treated with US four times in the same medium and supernatants were collected during 48 h of incubation of senescent cells (USS). (Fig. 3F) Bright field images show changes in morphology of SCs in USS collected from control cells treated with LFU. Senescent cells in normal growth medium were the control. (Fig. 3G) Graphs show increased proliferation of SCs in USS and (Fig. 3H) decreased area of ​​expansion in USS of senescent cells. Graphs are plotted by mean ± SD. A minimum of 200 cells were analyzed in the graphs (Fig. 3C) and (Fig. 3H). Scale bar = 300 μm. [Figure 4]Figures 4A-4D show that low frequency ultrasound reduces mitochondrial length and lysosomal intensity in senescent cells. (Figure 4A) Representative immunofluorescence images of mitochondrial morphology and lysosomal fluorescence in normal cells, senescent cells, and senescent cells treated with LFU stained with Mitotracker and Lysosomal Tracker. Scale bar = 10 µm. (Figure 4B) The ratio of lysosomal to mitochondrial staining intensity is reduced by ultrasound treatment of senescent cells. (Figure 4C) Quantification of mitochondrial length shows a decrease in length after LFU. Results are presented as mean ± SD, a minimum of 8 cells were analyzed for n>3 experiments, and significance was determined using a two-tailed unpaired t-test. ***p-value <0.001. (Figure 4D) Diagram of a working model for rejuvenation of senescent cells by activation of autophagy via LFU inhibition of mTORC1 activity. [Diagram 5] Figures 5A-5E show that reversal of replicative senescence by ultrasound allows the growth of a larger number of cells. (Figure 5A) Growth rates calculated as the number of cumulative cell population doublings (CPDs) for control HFFs and LFU-treated cells passaged every 48 h from P13 to P24 passages and treated at every other passage. (Figure 5B) Cells treated with LFU were smaller than p24 control cells and even p13 cells. (Figure 5C) The fraction of SA-β-galactosidase positive cells was decreased after LFU treatment. (Figure 5D) Similarly, LFU treatment of MSCs increased cell numbers at P10-19 treated at every other passage. (Figure 5E) MSCs treated with LFU showed normal differentiation into (ORO) adipocytes and (ARS) osteocytes. Alizarin Red S dye labeled osteogenesis (ARS) and Oil Red O dye labeled lipid droplets (ORO). Results are presented as mean ± SD, a minimum of 200 cells for spreading area and 150 cells for percentage β-galactosidase analysis was used, n > 3 experiments, significance was determined using a two-tailed unpaired t-test, ***p value < 0.001, **p value < 0.01, and *p value < 0.05. [Figure 6] FIG. 6 is a side view of an LFU device illustrating treatment of the foot using the present invention. [Figure 7]7A-7E show the effect of ultrasound treatment on the performance of aged mice. (Fig. 7A) Schematic of the treatment regimen. Mice (C57BL / 6J strain, 22-24 months old) were treated with LFU for 30 min every 3 days for 1 month and / or ran on the treadmill 12 times for 20 min. Mice were then rested for 1 month and treated or exercised for the second month. Each test group included 4 male and 4 female mice. (Fig. 7B) Graph of the results of the inverted cling test after the first month and (Fig. 7C) after the second month of treatment. (Fig. 7D) Results of the treadmill test after the first month and (Fig. 7E) results of the treadmill test after the second month of treatment. Groups were untreated (sham), treated with LFU (US), treated with exercise (EX), treated with rapamycin (RAP), treated with LFU + exercise (EXUS), and treated with rapamycin + LFU (RUS). Results were plotted as mean ± SD. Statistical significance was determined using Student's t-test. P values ​​> 0.05 are represented by ns. Statistical significance is provided by p value. *p value < 0.05, **p value < 0.001, and ***p value < 0.0001. [Figure 8]Figures 8A-8E show that LFU significantly reduces the fraction of senescent cells in kidney and pancreas. Mice aged 22-25 months were treated with LFU at 1x power every day (LFU_D1), every other day (LFU_D2), every third day (LFU_D3), and every day at 1.3x power (LFU_1,3_D1). After two rounds of 2-week treatment and evaluation, mice were euthanized and kidneys and pancreas were collected for SA-β-galactosidase staining. (Figure 8A) Diagram of the experiment showing the sequence of events. (Figure 8B) Kidney sections stained with SA-β-galactosidase from sham-treated and LFU_D3 mice. Under a color camera, β-galactosidase staining appeared blue, and the majority of cells in the kidneys of sham-treated mice were stained, but not in the kidneys of LFU_D3. (FIG. 8C) SA-β-galactosidase stained images of pancreatic sections indicated by color. Scale bar=150 μm and 20 μm. (FIG. 8D) Quantification of β-galactosidase stained area of ​​kidney sections. (FIG. 8D) Quantification of β staining of pancreatic sections. β-galactosidase staining was significantly reduced in all LFU treated mice. Results are plotted as mean ± SD. Statistical significance was determined using Student's t-test. P values ​​>0.05 are represented by ns. Statistical significance is provided by p value. *p value <0.05, **p value <0.001, and ***p value <0.0001 (n=10 mice). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description of the Invention While making and using various embodiments of the invention are discussed in detail below, it is understood that the invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely examples of specific ways to make and use the invention and do not delimit the scope of the invention.

[0015] To facilitate understanding of the present invention, a number of terms are defined below. Terms defined herein have the meanings commonly understood by one of ordinary skill in the art in the areas relevant to the present invention. Terms such as "a," "an," and "the" are intended to represent a singular entity only, but also to include a general classification for which a specific example may be used for illustration. While the terminology herein is used to describe certain embodiments of the present invention, their use is not intended to limit the present invention, except as set forth in the claims.

[0016] Ultrasound rejuvenation differs from existing therapies that address aging, such as exercise, senolytics, and other drug therapies, in that it is non-invasive, has no known adverse side effects, and can treat the entire organism and even internal organs. There is evidence that both exercise and ultrasound act mechanically to reverse aging through similar biochemical pathways. The advantage of LFU is that it allows for more tissue rejuvenation by reaching tissues and organs when an individual is unable to exercise them. In the case of senolytics that cause selective apoptosis of senescent cells, they can reverse some aspects of aging in older mice. 5、6 These are currently under consideration for clinical trials to help the elderly. 7 Because senolytic agents involve biochemical agents, they are difficult to deliver locally and it is unclear how they will affect senescent cells from different tissues. Furthermore, there are concerns about the duration of senolytic treatment. 8,9 , the effector molecule may simultaneously have deleterious effects on other cell types. 3Senescent cell death also leaves gaps in tissues that require tissue growth to repair. It would be much better to rejuvenate senescent cells in situ to rapidly restore normal function. There are potentially other drugs or treatments that enhance senescent cell rejuvenation by enhancing the biochemical pathways involved. LFU could enhance these treatments because it can be flexibly applied to reinforce the changes required. Importantly, mechanical treatments, unlike drugs or other therapies, act through normal biochemical pathways, and ultrasound provides a flexible way to activate these pathways non-invasively. Because ultrasound treatments reverse the senescent phenotype without cell death and can be applied repeatedly, LFU treatments provide benefits over time to older individuals. LFU irradiation below the damage threshold - used here as the definition of "low power" - also allows for peak strains of peak pressures higher than those easily achievable by exercise, and acceleration forces several orders of magnitude greater than those achievable by healthy exercise regimes. LFU irradiation can reach nearly all tissue types, including tissues shielded by bone and bone itself. Thus, the LFU treatment regime is more universal than conventional exercise.

[0017] The present invention includes a non-invasive method of treating aging using low frequency ultrasound. LFU irradiation can cause mechanical stretching of cells even in the body of an organism, and reverse the characteristics of senescent cells to prevent the effects of senescent cells on the function of the tissue in which they reside. Reversal of senescence by LFU is at least one of activating cell proliferation, reducing cell size, increasing secretion of growth factors, increasing mitochondrial division, and / or promoting wound healing. LFU treatment improves the function of aged mice, specific organs, wound healing, and allows greater expansion of normal cells in vitro. Ultrasound can be delivered to the whole organism, or smaller targeted ultrasound devices can be made for specific organ or cell applications.

[0018] As used herein, the term "low frequency ultrasound (LFU)" refers to wavelengths on the order of centimeters and amplitudes-motions smaller than a few microns. In one example, the wavelengths are on the order of organ size and the amplitudes are on the order of a single cell. For example, repetitive low frequency ultrasound treatment can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900 kHz, 1 MHz, or more, but is often 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900 kHz, or 1 MHz. Repetitive low frequency ultrasound treatments can be 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1,000, 5,000, 10,000, but often <500 mW / cm 2 It could be.

[0019] As used herein, the term "treating" refers to inhibiting, preventing, curing, reversing, attenuating, mitigating, minimizing, suppressing, or stopping the deleterious effects of a disease, and / or causing a reduction, slowing, or regression of a disease. Those skilled in the art will appreciate that a variety of methods and assays can be used to assess the onset of a disease, and similarly, a variety of methods and assays can be used to assess the reduction, slowing, or regression of aging.

[0020] Mechanotherapy. In certain aspects of the present disclosure, mechanotherapy or ultrasound therapy is provided to create and provide cyclic forces to senescent cells of a subject in need thereof. Immersion mechanotherapy may include applying one or more programmed cycle waves to a subject in a homogeneous immersion liquid for a predetermined period of time. The programmed cycle waves impart cyclic forces with a controlled duration, magnitude, and frequency sufficient to deform target cells (e.g., senescent cells or tissues with senescent cells) such that a mechanically induced rejuvenation process is induced. The created cyclic forces are low in intensity and small enough that normal cells and healthy tissues that the cyclic forces affect do not suffer mechanical or thermal damage. From a physical perspective, ultrasound and kinetic cyclic forces appear to be significantly different, especially since the force vector during ultrasound treatment changes tens of thousands of times per second. However, the average stress and pressure exerted on the tissue are remarkably similar. If cellular processes cannot track the change in direction of the acoustic waves, the remaining force envelope corresponds to the intensity of the treatment.

[0021] In some examples, the frequency and magnitude of the periodic force may be predetermined based on the target tissue. A sequence of programmed cycle waves may be applied to a subject (e.g., the whole body of a patient, a part of a body of a patient) at low intensity and frequencies ranging from about 20 to 250 kHz for an extended period (e.g., several hours) to induce reversal of aging in target cells (e.g., senescent cells) while promoting or preserving the proliferation of normal cells of the treated subject. The programmed duty cycle of the waves can be varied on the order of a few seconds, and the inventors have tested modulations from 0.1 Hertz to 10 Hz. The frequency of the sound carrier waves described above ranges from 10 kHz to possibly 250 kHz for large tissue sections, but can be several MHz for localized areas. The periodic force applied to the subject (e.g., internal organs) mechanically deforms the target / normal cells such that a mechanically induced reversal of aging process is induced in the target cells. In some examples, the frequency and magnitude of the cyclic force, or duration of a treatment session may be determined based on the target disease and / or the part / area of ​​the body being treated (e.g., whole body, arm, leg, breast, etc.).

[0022] As used herein, the term "spontaneous cell death" or "apoptosis" includes, but is not limited to, apoptosis, autophagy, and certain forms of necrosis. In the present disclosure, spontaneous cell death can be caused by a sequence of cyclical repetitive forces that differ from cell death caused by increasing amplitude or intensity, such as traditional ablation procedures. Specifically, as used herein, the term "apoptosis" refers to a controlled series of biochemical events that result in cell suicide, and is characterized by easily observable morphological and biochemical phenomena, such as deoxyribonucleic acid (DNA) fragmentation, chromatin condensation, chromosome migration in the cell nucleus, formation of apoptotic bodies, mitochondrial swelling, etc.

[0023] The terms "subject", "individual", "user" and "patient" are used interchangeably herein and refer to a mammal, such as a vertebrate, preferably a human. Mammals include, but are not limited to, mice, monkeys, humans, livestock, sport animals and pets. Tissues, cells and progeny of those biological entities obtained in vivo or cultured in vitro are also included. In some cases, the subject may have senescence or aging.

[0024] The target cell for the method of the present disclosure can be any cell that requires treatment for aging, senescence, or wound healing. In some embodiments, the target cell is a senescent cell. The target cell can include senescent cells in different regions of the patient's body. Senescent cells that can be used in the method of the present disclosure include, but are not limited to, prostate cells, breast cells, colon cells, lung cells, head and neck cells, brain cells, bladder cells, white blood cells, ovarian cells, kidney & testicular cells, melanocytes, liver cells, cervical cells, pancreatic cells, or gastrointestinal cells. In some examples, the target cell can be a cell of a tissue in a disease state where regeneration, proliferation, repair, etc. are desired.

[0025] In some embodiments, mechanotherapy may induce or control apoptosis of target cells by exposing them to cyclic stretch / pressure forces with predefined characteristics. In some embodiments, the disclosed methods at least partially stimulate, increase, release, activate, promote, enhance activation, sensitize, or upregulate apoptosis signaling pathways in tumor cells. Mechanotherapy may effectively activate cell surface receptors involved in apoptosis signaling pathways in tumor cells. The cell surface receptors may be capable of sensing mechanical cues, irradiation, or waves. Immersion mechanotherapy may effectively modify tumor cells and simultaneously affect senescent cells by applying cyclic and / or structured remote forces, such as ultrasound, to target tissues.

[0026] The immersion mechanotherapy of the present invention does not induce spontaneous cell death of normal cells. As used herein, the term "normal cells" refers to essentially healthy cells that have normal functions to maintain the correct functioning of tissues, organs, and organ systems. Normal cells may undergo spontaneous cell death as part of normal development to maintain tissue homeostasis and in response to irreparable damage. One or more cycle waves may be applied to normal cells in a target tissue without inducing spontaneous cell death of normal cells.

[0027] Various characteristics of the periodic force, such as intensity, frequency, amplitude, etc., may represent the intensity, frequency, or amplitude levels at the effective tissue site or the force delivered to the target cells. As an example, the force may include a low intensity cycle applied to the tissue site for several hours. In some examples, the periodic force applied directly to the tissue site or to the target cells may include long exposure times (e.g., several hours), low frequencies (e.g., 5-30 kHz, 30-250 kHz, 150 kHz-1 MHz, etc.) and low intensity levels (e.g., 5, 10, 15, 20, 25, 40, 50, 60, 70, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, <500 mW / cm 2 , and ranges therebetween). Periodic forces are quite generally wave mediated and vary based on their waveform or structured waveform, etc., which are used interchangeably unless the context suggests otherwise. Periodic forces can be transmitted to a subject while the subject is immersed in a liquid contained in a tub, which may also include water that is heated. As used herein, "periodic force" refers to a force envelope that is recurring or repetitive, and is manifested as a modulation of the force envelope with an underlying pressure.

[0028] The waveform can be any shape, so long as it achieves uniform or reasonably uniform membrane stress or stress envelope on the target cell / normal cell. In many cases, the waveform can be approximated to a sine wave. When such ultrasound is applied to the target cell / normal cell, the shape of the cell changes from a horizontal ellipsoid shape to a vertical shape, and then bounces periodically while the stress on the membrane remains relatively constant when the frequency and amplitude are matched. In some cases, the waveform can be determined based on the type of target cell. For example, the waveform delivered to the target tissue can be different for different types of cells or tissues to be treated. In some cases, the waveform delivered to the target tissue can depend on the mechanical properties of the target cell (e.g., cell kinetics and motility properties, diameter, stiffness, or inertia).

[0029] The waves applied to the subject may or may not be direction sensitive. Ultrasound in an optimal frequency range at a predefined amplitude may expose the target / normal cells to periodic deformations due to the same modest force acceleration and acoustic pressure. For example, when ultrasound is applied to a cell at a relatively low frequency (e.g., 20-250 kHz), the shape of the cell may change from a horizontal ellipsoidal shape to a vertical shape, and then periodically bounce while the stress on the membrane remains relatively constant. This type of rapid deformation may also be referred to as an extensional force, since it has no specific direction other than the periphery of the cell. This advantageously allows for a uniform immersed treatment that does not require precise placement or orientation of the ultrasound device relative to the subject. For example, for any part of the subject immersed in the liquid of the tub, the ultrasound given will not induce side effects in normal cells, while the effectiveness of the treatment may be substantially the same.

[0030] In some cases, the waves delivered to and experienced at the site of the target tissue or target cells may be in the low intensity range, since the energy absorbed by the subject is a small fraction of the energy emitted by the transducer. For example, low intensity may be in the range of 450 mW / cm 2 , 400mW / cm 2 , 350mW / cm 2 , 300mW / cm 2 , 250mW / cm 2, 200mW / cm 2 , 150mW / cm 2 , 100mW / cm 2 , 90mW / cm 2 , 80mW / cm 2 , 75mW / cm 2 , 70mW / cm 2 , 60mW / cm 2 , 50mW / cm 2 , 40mW / cm 2 , 30mW / cm 2 , 25mW / cm 2 , 20mW / cm 2 , 15mW / cm 2 , 10mW / cm 2 or less than 450mW / cm 2 or less than 10mW / cm 2 By low intensity, we mean 100 mW / cm, which we believe is harmless to almost all tissues except the fetus and the cornea. 2 This refers to the average absorption limit of common diagnoses.

[0031] In some cases, the acoustic waves at the site of the target tissue or target cells may have an optimal range of frequencies based on the mechanical properties of the target cells. For example, the frequencies may be from about 5 kHz to 30 kHz, 30 kHz to about 100 kHz, 30 kHz to about 150 kHz, 30 kHz to about 200 kHz, 30 kHz to about 250 kHz, 40 kHz to about 100 kHz, 40 kHz to about 150 kHz, 40 kHz to about 200 kHz, 40 kHz to about 250 kHz, 50 kHz to about 100 kHz, 50 kHz to about 150 kHz, 50 kHz to about 200 kHz, 50 kHz to about 250 kHz, 60 kHz to about 100 kHz, 60 kHz to about 150 kHz, 60 kHz to about 200 kHz, 60 kHz to about 250 kHz, 70 kHz to about 100 kHz, The ultrasonic frequency may be in the range of 70 kHz to about 150 kHz, 70 kHz to about 200 kHz, 70 kHz to about 250 kHz, 30 kHz to about 210 kHz, 30 kHz to about 220 kHz, 30 kHz to about 230 kHz, 30 kHz to about 240 kHz, 40 kHz to about 210 kHz, 40 kHz to about 220 kHz, 40 kHz to about 230 kHz, 40 kHz to about 240 kHz, 50 kHz to about 210 kHz, 50 kHz to about 220 kHz, 50 kHz to about 230 kHz, or 50 kHz to about 240 kHz, 150 kHz to 1 MHz, and levels within these recited frequencies.

[0032] In some embodiments of the present disclosure, the energy may be delivered by modulating it. In some examples, the amplitude of the sound is sufficient to induce a certain amount of deformation of the shape of the cells without introducing mechanical damage to the target cells or normal cells. For example, the shape of the target cells may be deformed by about 1% to about 5%, about 3% to about 8%, about 5% to about 10% when irradiated. For example, the on-off ratio of irradiation for a given time interval - called the duty cycle - may be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc. The duty cycle is defined as the fraction of time that the signal is "on" (e.g., transmitted) per unit of time. The intensity or amplitude of the wave is determined such that thermal damage is not introduced to the target tissue or subject.

[0033] The cyclical force may be applied to the target tissue or cells for a period of time. This period of time may be a range of time. For example, in immersion therapy, the waveform may be delivered to the subject as a treatment session for about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 24 hours, 48 ​​hours, or more. The treatment session may be an exposure to radiation that is continuous or intermittent. The treatment session may include one or more sub-sessions that may or may not utilize the same cyclical force or waveform. In some examples, the treatment may be repeated for the same or different lengths of time, one or more times over days, weeks, months, years, or the lifetime of the subject. The treatment may be performed continuously while the subject is sitting in a hot tub. Alternatively or additionally, the treatment may be repeated at a predetermined time interval between them.

[0034] As used herein, wave characteristics such as intensity, amplitude, and frequency refer to the intensity, amplitude, and frequency levels at the effective tissue site, not the actual output values ​​of the ultrasound transducer. In some cases, one or more characteristics of the wave as a direct output from the ultrasound transducer may differ from the characteristics of the periodic force effective at the target tissue or target region.

[0035] The output of the ultrasonic transducer to create such waveforms may have a higher intensity level than the effective amount resulting at the target tissue site to account for energy loss or dispersion during transmission. For example, while the ultrasonic waves traverse the tissue and liquid before reaching the target tissue / area, a certain amount of energy is absorbed or scattered by the biological tissue (e.g., skin, bone, muscle, and underlying fascia) and the liquid in the tub. Due to the low frequency characteristics, the loss of intensity may be less and the penetration depth may be longer. As an example, 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 65% or less of the energy may be absorbed during the transmission of the wave. This advantageously allows for immersion mechanotherapy as the waves can be effectively transmitted to the internal tissues of an individual through the pool / container of liquid and surrounding organs. Furthermore, due to the low frequency and low intensity characteristics of the waves, the heat generated as a result of the waves penetrating tissue was calculated to be insignificant based on experimental values ​​of heat vs. power at these wavelengths.

[0036] The waves may act on the subject in the form of immersion mechanotherapy. In some embodiments, at least a portion of the subject's body is immersed in the liquid contained in the tub / container. Such a portion of the subject's body may be, for example, 0.5% to 100% of the subject's body. In particular, the subject may have 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the body portion immersed in the liquid. The disclosed ultrasound waves may penetrate the portion or all of the body immersed in the liquid without inducing side effects or damage to normal cells / tissues.

[0037] Mechanotherapy Systems and Devices. In one aspect, the present disclosure provides devices and systems configured to perform the above-mentioned mechanotherapy on a subject in need thereof. In some examples, the device or system may be handheld or may be operated, held, and / or controlled by a robotic arm, and includes a vessel or container for containing a liquid in which at least a portion of the individual / subject is immersed, and one or more ultrasound transducers capable of generating one or more programmed cycles of waves for a determined period of time to treat senescent cells. The vessel may be sized and shaped to hold one or more individuals. In some examples, the vessel may include an internal shape or configuration to facilitate the transmission of waves to the individual.

[0038] The present invention can use one or more ultrasound transducers capable of generating one or more programmed outputs for a predetermined period of time to treat an individual. The disease can be a wound in an individual. The one or more transducers can be configured to create structured waves as described elsewhere herein. The structured waves can be focused or unfocused. The ultrasound transducer can be a single element or array of ultrasound transducers, or a composite assembly of emitters.

[0039] In one example, the one or more transducers may be an array of ultrasonic transducers. The array of ultrasonic transducers may be directed to one or more tissues in the body. In some examples, multiple ultrasonic transducers may be mounted at the sidewall and / or bottom cavity of the container. In some embodiments, the one or more transducers may be moved to the interior space of the container using a waveguide. In some embodiments, the one or more transducers may be moved to the outside of the container. In some embodiments, the one or more transducers may be placed near the bottom wall of the container. In some embodiments, the one or more transducers may be placed near the sidewall of the container. In some embodiments, the one or more transducers may be immersed in the liquid of the container. The one or more transducers may be placed in a desired location (e.g., in optimal space, direction relative to each other) so that when the transducers are activated simultaneously, ultrasonic waves can be collectively created to achieve a desired effect (e.g., direction, focal plane, intensity, etc.).

[0040] In some embodiments, one or more transducers may be packaged and sealed in a panel for sterilization purposes. The panel may have a substantially smooth surface and may be composed of a material that can be sterilized by conventional methods compatible with the tab, such as steam, heat and pressure, chemicals, UV light, etc. In some embodiments, the surface of the panel may be disposable. The panel may be removably coupled to one or more transducers. In some examples, the panel may be composed of a material to reduce wave resistance and may include geometric features (e.g., thin walls or sheets) to reduce wave resistance. In some examples, the panel may include a docking feature or structure that mates with the shape of one or more transducers to provide a smooth fit.

[0041] The one or more ultrasonic transducers may or may not be in direct contact with the liquid. The one or more ultrasonic transducers may or may not be in direct contact with the bathtub. When the individual is placed, the individual may or may not be in direct contact with the one or more ultrasonic transducers.

[0042] The one or more ultrasound transducers may be removably coupled to an apparatus used to hold the subject, such as a chair, bed, or tub, and / or may be permanently fixed to the apparatus. In some examples, the one or more ultrasound transducers may be mounted on a wall. For example, a transducer array (such as a phased array) may be coupled to the wall of a bathtub, and the direction of the sound waves may be controlled using beamforming techniques.

[0043] The ultrasonic transducer or transducers may be hermetically sealed and water resistant. The ultrasonic transducer or transducers or ultrasonic device may be provided with an internal cooling system for stabilizing the operating temperature of the ultrasonic device or transducers. Any suitable cooling method for cooling the ultrasonic device may be utilized. The cooling method may be passive cooling, for example, by placing an ultrasonic probe thermally coupled to a heat sink or other cooling form (e.g., heat pipe, heat spreader, etc.). Passive cooling refers to the dissipation of heat from an ultrasonic transducer (e.g., ultrasonic probe) by thermal contact with a heat sink or cooling fins. In some examples, a coolant, such as a fluid or gas coolant, may be circulated over the surface of the ultrasonic transducer, cooling fins, and / or heat sink to assist passive cooling. The cooling method may be active cooling, for example, utilizing a thermoelectric cooler driven by a temperature controller to regulate or stabilize the ultrasonic transducer operating temperature.

[0044] In some embodiments, one or more transducers may be carried by a robotic arm. The robotic arm may be configured to provide one or more degrees of freedom of movement to the one or more transducers. The one or more transducers may be controlled to be positioned in a desired position or orientation, for example, so that different parts of the body can be treated. The position or orientation of the one or more transducers may be fixed during a treatment session. Alternatively or additionally, one or more transducers may be controlled to move during a treatment session (e.g., curve movement, positioned at different positions in different sub-sessions).

[0045] In some examples, the robotic arm may be a gantry. The robotic arm may be a six-axis robotic arm. The robotic arm may be capable of performing about one or more, two or more, three or more, four or more, five or more, or six or more axes of movement. The robotic arm may include one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more joints. The joints may include motors that may enable the various support members to move relative to one another. The robotic arm may include one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more support members. In one example, the first support member may bear the weight of the end effector. The second support member may bear the weight of the first support member and / or the end effector, etc. The motors may enable rotation of one or more support members relative to one another. One or more sliding mechanisms may be provided that may allow for lateral displacement. The robotic arm may have a range of free movement that may match or exceed the range of movement of a human arm. Ball and socket joints may or may not be used by the robotic arm.

[0046] In some embodiments, one or more transducers may be fixed to an end effector of a robotic arm. The position and orientation of the one or more transducers may be controlled by controlling the robotic arm. In some examples, the robotic arm may be controlled by a robotic controller. The robotic controller may be under the control of a controller that positions the one or more transducers. With respect to the control system, a cascade proportional integral derivative control (PID) may be used to control the attitude and velocity of the robotic arm. It should be noted that there are various control algorithms that may be used to control the gimbal or carrier system, including, but not limited to, ON-OFF, PID mode, feed-forward, adaptive, intelligent (Fuzzy logic, Neural network, Expert Systems and Genetic) control algorithms. In a particular control model, such as PID control, the control system may vary with different control objectives / output variables (e.g., angular velocity, angular position, angular acceleration, or torque) and different input variables (e.g., input voltage). Thus, the control parameters may be expressed in various ways.

[0047] In some examples, the robotic arm controller may be configured to control the robotic arm using sensor data as feedback information. The sensor data may relate to the position of the end effector (i.e., one or more transducers) relative to the object. In some examples, sensors such as proximity sensors or imaging sensors may be used to provide such position / proximity information. In some examples, one or more ultrasonic transducers along with a receiver may be provided as ultrasonic sensors to determine proximity. In some examples, additional sensors may be included to provide such information.

[0048] In some examples, the robotic arm may automatically position one or more transducers in an initial position. In some embodiments, the robotic arm may be passively moved by a user. In such examples, the user pushes the arm to any position and the arm moves accordingly. The robotic arm may also be controlled in a compliant mode to improve human-robot interaction. For example, the compliant motion control of the robotic arm may use a collision prevention strategy and the position-force control may be designed to reduce unnecessary energy consumption while reducing the effects of possible collisions. Multiple ultrasonic transducers may be collectively actuated to create a series of force pulses. The number of transducers may be any number, such as numbers from 1 to 1000, and may emit with different amplitudes and / or phase relationships. For example, adjacent transducers may have a constant progressive phase shift or a variable phase shift to adjust the direction of the beam / wave. In some examples, the frequencies of the transducers may all be the same or may emit different frequency ranges to provide a composite waveform containing multiple frequency components.

[0049] Ultrasound may be focused, unfocused, or a combination of both. In some cases, additional components such as acoustic lenses or reflectors may be utilized to produce focused ultrasound. The focal plane or focal length of the transducer (array) may be adjusted to direct the beam to the location of the target area of ​​interest. In some cases, the ultrasound device may include an array of individually controlled transducers that allow steering and focusing of the beam. Beamforming techniques, such as phased array beamforming or beam control methods, such as the use of mirrors that move the acoustic lens to adjust the focal length of the device, may be utilized. The array of transducers may act collectively to create and transmit waveforms to the target location / area. In addition to the focal length of the ultrasound device, one or more characteristics of the wave, such as frequency, duty factor, amplitude, intensity, etc., may be modulated by controlling the array of ultrasound transducers.

[0050] Alternatively or additionally, the ultrasound may be unfocused. Unfocused ultrasound may pass through liquid and / or biological tissue immersed therein. Unfocused ultrasound may be applied to a large area / part of a subject. In some embodiments, one or more transducers may be configurable such that the provided ultrasound system may be able to switch between focused ultrasound mode and unfocused mode or may be able to operate in dual mode.

[0051] In some examples, one or more transducers may be customized to be neutral generators to provide additional user safety. For example, when using transducers or antenna arrays where the sum of the net voltages delivered is zero or near zero, safe human contact may be provided even if insulation fails, since liquid intrusion must pass through a zero volt contact before reaching any part of the interior of the transducer. Arrays of antenna elements may be constructed with transducers with alternating coil orientation or inverted piezoelectric crystals to allow for zero net voltage. Such alternating coil orientation or inverted piezoelectric crystal designs may be applied to one or more pairs of antenna elements or to the entire antenna array.

[0052] In some embodiments, the liquid in the tub may be controlled at a temperature of 4-45°C, 16-20°C, 20-40°C, 36-40°C, or other temperature range based on user preference. In some embodiments of the present disclosure, immersion mechanotherapy is delivered in the form of a tub or spa and may involve a heater to raise the water temperature to a level typically in the range of about 95°F. The transducer system may be part of the heating device. In some embodiments, the system may include a temperature control system. The temperature control system may include one or more temperature sensors and / or a temperature controller to control the temperature of the medium (e.g., liquid) as needed. The one or more temperature sensors may be positioned at any suitable location relative to the bathtub. The temperature may be manually adjusted by an individual, user, operator, or automatically controlled by a preprogrammed treatment plan.

[0053] In some embodiments of the present disclosure, the system may also include features for cooling overheated media or bath liquid. For example, water drainage or recycling may be used to keep the water temperature stable. Such features may be the same as known in conventional spa or massage equipment. For example, the water may be filtered and heated and recycled as is conventional in a spa via one or more hydrotherapy massage jet nozzles mounted in spaced locations on the sidewall. Care must be taken to avoid air bubbles, as they will absorb the ultrasound and render the system ineffective.

[0054] The medium that may be used in the mechanotherapy of the present disclosure may be any medium capable of transmitting the ultrasound waves of the present disclosure. In some embodiments, the medium is a liquid. In some embodiments, the medium is a gel. In some embodiments, the medium is water. In some embodiments, the medium is alcohol or saline.

[0055] In some embodiments, the liquid may be water. The liquid may be any suitable liquid that is safe for human contact and has an acoustic impedance similar to human tissue. For example, the acoustic impedance may be similar to or higher than that of the tissue / skin of the subject being treated. In some examples, chemicals may be added to the water. The chemicals that may be used in the medium may be any chemical that does not interfere with the wave transmission of the present disclosure. In some examples, the chemical is a salt. In some examples, chemicals such as chlorine may be periodically added to the spa water in a prescribed amount suitable for preventing the growth of bacterial organisms to maintain the water in a sanitary condition. Other chemicals, such as germicides, oxidizers such as chlorine, may also be periodically added to the water.

[0056] In some embodiments, a portion of the subject's body is submerged in the liquid of the tub. The portion of the subject's body may be, for example, 0.5% to 100% of the subject's body. In particular, the subject may have 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the portion of the body submerged in the liquid.

[0057] In some embodiments, the container or tub may include an open-top enclosure including a bottom wall and one or more side walls. In some embodiments, the container may include a bottom wall, opposing side walls, and opposing end walls. In some embodiments, the container may further include an operable top lid. The bottom wall and / or side walls of the container may be of any suitable shape. For example, the shape of the bottom wall or side walls of the container may include, but is not limited to, a circle, an ellipse, a rectangle, a square, a trapezoid, a triangle, or an irregular shape. In some embodiments, the inner wall of the container may be formed to reflect or have a predetermined geometric configuration to reflect ultrasound waves to an immersed part of the subject or a target location. In some examples, the inner wall of the container may be formed to modulate the wave field or may be composed of a US absorbing material that modulates the wave field so that undesired waves (e.g., waves reflected on the inner wall) can be reduced. This allows for controlled waves of desired frequency and intensity that are preferably received and effective in the immersed body. In some embodiments, the vessel may be selected from a tub, a bucket, a tank, a container, and a pool. In some embodiments, the vessel may be a tub. In some embodiments, the vessel is a spa tub. In some embodiments, the vessel is a swimming pool. The tank material may be any suitable type of material, including, but not limited to, glass, metal, or ceramic, aluminum and steel, fiberglass, plywood, porcelain, and the like. In some embodiments, the submerged portion material may be a high temperature ceramic.

[0058] In some embodiments, the system may further include a user positioning system. In some examples, the user positioning system may utilize a proximity sensor to detect the position of the user's body. The proximity sensor may be an ultrasound device in which an ultrasound transducer may be paired with one or more receivers to measure distance based on time of flight. Alternatively or additionally, additional sensors may be used to locate the subject's body. For example, additional proximity sensors (e.g., ultrasound sensors, cameras) may be used to detect the subject's position relative to the container / transducer or the subject's proximity to the transducer.

[0059] The controller may control one or more ultrasound transducers coupled to the container. The system may further include a computer system and one or more databases operably coupled to the controller over a network. The computer system may include a treatment planning module that implements the methods provided herein for generating a treatment plan.

[0060] The computer system may be used to create an individualized treatment plan based on personal / user information, device setup, diagnostic information, etc. Although the illustrated table shows the controller and computer system as separate components, the controller and computer system may be integrated into a single component.

[0061] For example, the treatment plan may include information about the level of mechanotherapy (e.g., ultrasound frequency, intensity, amplitude, duty cycle), type of treatment (e.g., senescent cell reduction, cellular senescence reduction, or wound healing), information about the treatment area (e.g., location, volume, tissue type, etc.), operating conditions (e.g., temperature control, focused beam / unfocused beam), treatment duration, user information (e.g., user's preferred spa temperature), or others.

[0062] The treatment plan may be created in a fully automated format, a semi-automated format, or a manual format. In some examples, the treatment plan may be created automatically after receiving diagnostic input or user information. For example, the frequency, amplitude, and intensity of the delivered ultrasound may be automatically determined based on diagnostic information (e.g., tissue location, volume, disease type, purpose of application) and / or user information. In some examples, the treatment plan may be created using AI techniques and / or machine learning methods. For example, a machine learning model may be trained to create the treatment plan. In some examples, the input data provided to the machine learning model may include diagnostic information, device information, personal information, or other information described elsewhere herein. In some examples, the output of the machine learning model may be a treatment plan or one or more treatment parameters (e.g., force characteristics, device setup, spa duration, etc.). The treatment plan may be dynamically adapted to real-time conditions based on feedback information. Alternatively or additionally, the treatment plan may be performed throughout without real-time feedback information.

[0063] In some examples, the machine learning methods used to generate the treatment plan may include one or more machine learning algorithms. Examples of machine learning algorithms may include support vector machines (SVMs), naive Bayes classification, random forests, deep learning models, feedforward neural networks, RBF networks, recurrent neural networks, convolutional neural networks, deep residual learning networks, or other supervised or unsupervised learning algorithms.

[0064] The controller may operate to provide information about pulse sequences to the ultrasound device's controller and / or manage the operation of the entire system through an installed software program. In some examples, the controller may also function as an element instructing the subject / user to perform a task, such as placing a body part in a predetermined position in a container, through a voice message generated using automated voice synthesis technology. The controller may receive commands from an operator instructing the mechanotherapy to be performed. Alternatively, the system may be for use at home, and the user may receive instructions through a user interface (e.g., a mobile application) operably linked to a computer system via a network. The controller is configured to operate the components of the system to perform a desired wave or cyclic force sequence, and may include various components such as a pulse generator module that generates data representing the timing, strength, and shape of the waves or ultrasound pulses produced, as well as the direction of the beam. In some examples, the controller may control the pulse generator module and / or a set of gradient amplifiers of the transducer to control the frequency, amplitude, and shape of the pulses or waves produced during the treatment. In some examples, the controller may control the phase shift of a phased array transducer to adjust the direction, focusing, and other characteristics of the ultrasound beam. In some examples, the controller may control the orientation and position of one or more transducers relative to the subject by controlling a robotic arm that carries the one or more transducers.

[0065] In some circumstances, the controller may also receive real-time patient data from a physiological acquisition controller that receives signals from sensors attached to the patient, such as ECG (electrocardiogram signals) from electrodes or respiratory signals from the lungs. The controller may be coupled to various sensors for monitoring the condition of the patient (e.g., wound healing progress), ultrasound transducers, and containers (e.g., liquid filling, liquid temperature, etc.). For example, a temperature sensor may be coupled to the controller for temperature control during operation. In some examples, the system may include a user positioning system that may receive commands to move the user to a desired position for treatment or to instruct the user to immerse a particular body part in a bathtub of water.

[0066] In some examples, the controller may include or be coupled to an operator console (not shown), which may include an input device (e.g., a keyboard) and a control panel and display. In a home use situation, the operator console may be a user interface. For example, the controller may have input / output (I / O) ports connected to I / O devices such as a display, a keyboard, and a printer. In some examples, the operator console may communicate over a network with a computer system that allows the operator to control the treatment procedure or modify the treatment plan on the screen of the display. In some examples, the user may be able to view the progression of a disease, such as the progression of wound healing, on the display.

[0067] The system may include a user interface. The user interface may be configured to receive user input and output information to a user. User input may relate to controlling a treatment procedure (e.g. wound healing, tissue recruitment, tissue regeneration, etc.), creating / modifying a treatment plan (e.g. selecting a body part to be treated), controlling a spa condition (e.g. temperature, massage mode, etc.), etc. User input may relate to vessel operation (e.g. massage mode, e.g. treatment level, water temperature, etc.), ultrasound operation (e.g. parameters for controlling waves delivered to a target area, such as frequency, amplitude, duration, etc.). User input may relate to various actions or conditions for creating a treatment plan. The user interface may be provided on a screen, such as a touch screen, or any other user-interactive external device, such as a handheld controller, mouse, joystick, keyboard, trackball, touch pad, button, verbal command, gesture recognition, posture sensor, thermal sensor, touch-capacitive sensor, foot switch, or any other device, or may be implemented via an app or software package.

[0068] The system may include computer systems and database systems, which may interact with or form the controller. The computer systems may include laptop computers, desktop computers, central servers, distributed computing systems, etc. The processor may be a hardware processor, such as a central processing unit (CPU), a graphics processing unit (GPU), a general-purpose processing unit (GPPU) which may be a single-core processor or a multi-core processor, multiple processors for parallel processing in the form of fine-grained spatial architecture, such as a field programmable gate array (FPGA), an application-specific integrated circuit (ASIC), a digital signal processor (DSP), and / or one or more embedded processors. The processor may be any suitable integrated circuit, such as a computing platform or microprocessor, a logic device, etc. Although the present disclosure is described with respect to a processor, other types of integrated circuits and logic devices are also applicable, especially dedicated processors for different functions. The processor or machine is not limited in data operation capacity. The processor or machine may perform 512-bit, 256-bit, 128-bit, 64-bit, 32-bit, or 16-bit data operations.

[0069] The system may include one or more databases. The one or more databases may utilize any suitable database technology. For example, a Structured Query Language (SQL) or "NoSQL" database may be utilized to store diagnostic data, e.g., image data obtained by an appropriate imaging modality, training data sets or trained models to generate treatment plans, treatment plan parameters, previous treatment plans, user preferred spa conditions, and the like. Parts of the database may be implemented using various standard data structures, e.g., arrays, hashes, (linked) lists, structures, structured text files (e.g., XML), tables, JSON, NOSQL, and the like. Such data structures may be stored in memory and / or in (structured) files. In another alternative, an object-oriented database may be used. An object database may contain many object sets grouped and / or connected by common attributes; these may be related to other object sets by some common attribute. An object-oriented database performs similarly to an associative database, except that objects are not simply pieces of data, but may have other types of functionality encapsulated within a given object. When the database of the present disclosure is implemented as a dedicated system, the use of the database of the present disclosure may be integrated into another component, such as a component of the present invention. Similarly, the database may map a data structure mix, such as objects and related structures. The database may be centralized and / or distributed in implementation. Portions of the data in the database, such as tables, may be exported and / or imported, and thus distributed and / or consolidated / migrated.

[0070] A network may establish connections between components in a system and / or to systems external to the system. A network may include any combination of local area and / or wide area networks using both wireless and / or wired communication systems. For example, a network may include the Internet and cellular networks. In one embodiment, a network uses standard communication technologies and / or protocols. Thus, a network may include links using technologies such as Ethernet, 802.11, worldwide interoperability for microwave access (WiMAX), 2G / 3G / 4G mobile communication protocols, asynchronous transfer mode (ATM), InfiniBand, PCI Express Advanced Switching, etc. Other networking protocols used on the network may include Multiprotocol Label Switching (MPLS), Transmission Control Protocol / Internet Protocol (TCP / IP), User Datagram Protocol (UDP), Hypertext Transfer Protocol (HTTP), Simple Mail Transfer Protocol (SMTP), File Transfer Protocol (FTP), etc. Data exchanged over the network may be represented using technologies and / or formats including binary image data (e.g., Portable Network Graphics (PNG)), HyperText Markup Language (HTML), Extensible Markup Language (XML), and the like. Additionally, all or some links may be encrypted using conventional encryption techniques, such as secure sockets layers (SSL), Transport Layer Security (TLS), Internet Protocol Security (IPsec), and the like. In alternative embodiments, entities on the network may use custom and / or proprietary data communications instead of or in addition to those described above.

[0071] Example 1. Induction and characterization of senescence

[0072] To test the generality of any senescence treatment, we used four different methods to induce senescence in Vero cells: replicative, hydrogen peroxide, sodium butyrate, and bleomycin sulfate. Senescence was measured as a decrease in growth rate, an increase in β-gal activity, an increase in cell size, and secretion of a proinflammatory factor called SASP. All four criteria were consistent with each type of senescent cell (Figures 1A-1G). In each of these cases, cell growth was effectively shut off, with minimal remaining growth (Figure 1B). There was a significant difference in the size of the senescent cells, where cells treated with bleomycin sulfate were the largest and replicative senescent cells were the smallest (Figures 1C and 1D). To assay for SASP activity, we cultured the senescent cells overnight and collected the supernatant from each culture dish. The supernatant was diluted 1:1 with fresh medium and added to normal control cells. Changes in normal cell numbers were measured after 48 hours of incubation. Supernatants from senescent cells inhibited the growth of normal cells, as predicted by SASP (Figure 1E). When levels of β-galactosidase were measured by antibody staining, a relatively uniform increase in levels was observed (Figures 1F and 1G). Thus, by all four criteria, the four different types of senescent cells were indeed senescent.

[0073] Figures 1A-1G show the characterization of senescent cells (Figure 1A). 2 O 2、 Brightfield images of senescent cells induced by sodium butyrate (SB), doxorubicin (Dox), and bleomycin sulfate (BS). Scale bar = 300 μm. (Figure 1B) Quantification of proliferation shows no senescent growth after 48 h of incubation. (Figure 1C) Senescent cells are larger compared to control cells. (Figure 1D) Quantification of average cell volume of cells treated with bleomycin sulfate compared to control cells. (Figure 1E) Conditioned medium from senescent cells inhibits the growth of normal proliferating cells. (Figure 1F) SA-β-galactosidase staining of control (proliferating) and BS-treated senescent cells. Scale bar = 300 μm. (Figure 1G) H 2 O 2and levels of the β-galactosidase senescence marker in SCs induced by SB. Scale bar = 300 μm. Data from a minimum of 150 cells (FIGS. 1B-1H) were analyzed for spreading area and cell volume from n=2 experiments.

[0074] Example 2. Low frequency ultrasound (LFU) irradiation activates the reversal of growth and aging phenotypes.

[0075] To determine whether mechanical activity could reverse some of the phenotypes of senescent cells, we irradiated senescent cells with low-frequency ultrasound at low-to-medium power levels and a modulation frequency of about 1 Hz, with a duty cycle of 50%. After 20 min of irradiation at 30 kHz, the cells were cultured for 48 h.

[0076] Figures 2A-2G show that LFU reverses cellular senescence. (Figure 2A) Schematic of senescence reversal experiment. SB-treated Vero cells were treated with LFU and passaged every 48 hours for 8-10 days. (Figure 2B) Graph showing cell growth as fold change over 48 hours. Treated cells were passaged every 48 hours from P0 to P3. (Figure 2C) Cell area of ​​LFU-treated senescent cells largely recovers to normal by P3. (Figure 2D) Representative anti-p21 immunofluorescence images of P3 control and LFU-treated cells. Scale bar = 300 μm. (Figure 2E) Quantification of fluorescence intensity of control and LFU-treated P3 cells stained with p21. Shown as mean ± SD for >200 cells in each condition. (Figure 2F) LFU increased proliferation as determined by EDU staining. (Figure 2G) Quantification of EDU-positive P3 cells. All graphs plotted by mean ± SD. A minimum of 200 cells were analyzed for each condition.

[0077] Regarding cell growth, the cells continued to grow for days. The growth rate did not decrease, so the cells appeared to have returned to normal. When cell size increased, ultrasound treatment caused an increase in the fraction of small cells that significantly overlapped in size with normal cells. After 12 days of growth, the fraction of small cells increased, representing a slower growth rate of larger cells. Regarding SASP secretion, the cells first produced a supernatant that significantly inhibited the growth of control cells and caused a significant increase in cell size; this led us to test whether SASP secretion ceased after LFU (illustrated in FIG. 3A). Supernatants collected after LFU treatment had no effect on growth or spreading area compared to supernatants collected from the same cells before LFU treatment (FIGS. 3B-3D). Furthermore, we also checked for wound healing by cell scraping in the presence of supernatants from senescent cells and treated senescent cells. Supernatants from senescent cells inhibited wound healing, whereas supernatants from cells treated with ultrasound did not. Thus, reversal of the senescent cell state by ultrasound may be partial; however, rejuvenated cells may dominate over time and essentially provide a restored population of normal cells.

[0078] Example 3. LFU irradiation activates normal cells to secrete growth promoting factors.

[0079] Many studies have shown that physical exercise slows down tissue and brain aging. To test the possibility that LFU provides benefits to normal cells and their senescent neighbors, we treated normal cells with LFU for 3 days (1 hour per day) and then collected the supernatant (Figure 3E). The supernatant after this period was added to senescent Vero cells. Surprisingly, senescent cells were rejuvenated by a 1:1 mixture of USS (sonicated normal cell secretion) medium and normal medium. USS activated growth (Figure 3F) and caused a decrease in the spreading area of ​​senescent cells (Figures 3F and 3G). This indicated that ultrasound treatment could stimulate the secretion of growth molecules from normal cells, which would further benefit tissues in which a mixture of normal and senescent cells exist. Thus, there is every indication that ultrasound treatment provides benefits to tissues of aged organisms in which a mixture of senescent and normal cells is expected to exist.

[0080] Figures 3A-3H show that USS inhibits control but not control growth of senescent cells. (Figure 3A) Schematic of the experiment showing that LFU blocks SASP secretion. SCs were cultured in growth medium for 24 h and then treated with LFU for 30 min. Supernatants were collected after LFU treatment (S0) and again after another 24 h incubation (S24). To confirm the effect of LFU treatment, supernatants S0 and S24 were used to confirm the growth of non-senescent control cells. (Figure 3B) Representative brightfield images of control cells after 48 h incubation in normal growth medium, S0, and S24. Quantification of cell number (Figure 3C) and cell area (Figure 3D) after 48 h in control, S0, or S24 supernatants. (Figure 3E) Schematic timeline and strategy of LFU treatment of normal proliferating cells. Schematic diagram showing that control cells were treated with US four times in the same medium and supernatants were collected during 48 h of incubation of senescent cells (USS). (Fig. 3F) Bright field images are collected from control cells treated with LFU and show changes in morphology of senescent cells (SC) in the supernatant after 48 h in response to USS. Senescent cells in normal growth medium were the control. (Fig. 3G) Graphs show that SC in USS increased growth and (Fig. 3H) decreased spreading area. Graphs are plotted by mean ± SD. A minimum of 200 cells were analyzed for graphs (Fig. 3C) and (Fig. 3H). Scale bar = 300 μm.

[0081] Example 4. LFU affects mitochondrial dynamics and lysosomal levels.

[0082] Since aging is associated with increased mitochondrial fusion, we followed up on the changes in mitochondrial length with aging and the reversal of aging with ultrasound. For comparison, we also checked the morphology of lysosomes and microtubules, since they are also known to change in the aging state. There was a significant increase in mitochondrial length and number of lysosomes in aging cells. Thus, we support the model outlined in Figure 4D and propose that ultrasound acts by increasing autophagy through the inhibition of mTORC1.

[0083] Figures 4A-4D show that LFU reduces mitochondrial length and lysosomal intensity in senescent cells. (Figure 4A) Representative immunofluorescence images of mitochondrial morphology and lysosomal fluorescence in normal cells, senescent cells, and senescent cells treated with LFU stained with Mitotracker and Lysosomal Tracker. Scale bar = 10 µm. (Figure 4B) The ratio of lysosomal to mitochondrial staining intensity is reduced by LFU treatment of senescent cells. (Figure 4C) Quantification of mitochondrial length shows a decrease in length after LFU. Results are presented as mean ± SD, a minimum of 8 cells were analyzed for n>3 experiments, and significance was determined using a two-tailed unpaired t-test. *** p value < 0.001. (Fig. 4D) Diagram of the working model for rejuvenation of senescent cells by activating autophagy via LFU inhibition of mTORC1 activity.

[0084] Example 5. Reversal of replicative senescence

[0085] Since replicative senescence likely reflects an inherent property of normal cells that limits growth, we tested whether ultrasound could effectively create an immortal cell population. Human foreskin fibroblasts after P13 passage showed a significant increase in the mean cell size, a decrease in growth rate, and an increase in β-galactosidase activity (Figure 5A-C). However, after LFU treatment, senescent cells grew like normal cells (Figure 5A). When cells were treated with LFU after each passage, they continued to grow beyond P24 passage without changing their growth rate (Figure 5A). Since the average time per cell division is 24-36 hours, this means that HFFs were able to grow more cells by rejuvenation with LFU. After 24 passages, LFU-treated cells stopped growing when cultured on a soft matrix, indicating that the cells still exhibit stiffness-dependent growth. Thus, reversal of senescence by ultrasound can effectively increase the replicative lifespan of cells without resulting in transformation or other obvious changes in cell phenotype.

[0086] To test whether mesenchymal stem cells could also be expanded without altering their phenotypic behavior, P10 mesenchymal stem cell cultures were treated with LFU before each passage until P19. They showed a significant 64-fold increase in growth compared to control cells (Figure 5D). However, when incubated in adipocyte differentiation medium, they adopted the phenotype of adipocytes, as revealed by Oil Red O staining. Alternatively, when incubated in osteocyte medium, they became osteocytes, as revealed by Alizarin Red S staining for calcium deposition. In both cases, cells treated with LFU showed much more differentiation than untreated controls (Figure 5E).

[0087] Figures 5A-5E show that reversal of replicative senescence by LFU increases cell number. (Figure 5A) Growth rate calculated as cumulative cell population doublings (CPD) of control HFF and LFU-treated HFF cells passaged every 48 hours from P13 to P24 passages and treated at every other passage. (Figure 5B) Cells treated with LFU were smaller than p24 control cells and even p13 cells. (Figure 5C) The number of SA-β-galactosidase positive cells decreased after LFU treatment. (Figure 5D) Similarly, LFU treatment of MSCs increased cell number at P10-19, where treatment was at every other passage. (Figure 5E) MSCs treated with LFU showed normal differentiation into (ORO) adipocytes and (ARS) osteocytes. Alizarin Red S staining dye labeled osteogenesis (ARS) and Oil Red O staining dye labeled lipid droplets (ORO). Results are presented as mean ± SD, a minimum of 200 cells for spreading area and 150 cells for percentage β-galactosidase analysis was used, n > 3 experiments, and significance was determined using a two-tailed unpaired t test. *** p-value < 0.001, ** p-value < 0.01, and * p-value < 0.05.

[0088] 6 is a side view of an US device showing the treatment of a foot using one type of the present invention. A transducer is shown sending sound waves to the foot. The sound waves may then contact a silencer. Those skilled in the art will understand that any part of the body, including any limb, torso, head, or entire body, may be exposed to sound waves in a manner similar to what we have described herein.

[0089] Example 6. LFU treatment of aged mice improves performance.

[0090] To determine whether LFU therapy is beneficial for aged organisms, we tested the effect of LFU therapy on aged mice by treating them for 30 minutes every 3 days. In these experiments, we found that the physical performance of mice treated with LFU and especially mice treated with LFU + exercise improved compared to the control group. The treatment schedule is illustrated in Figure 7A, showing that mice were treated for 30 minutes every 3 days for 1 month (all LFU groups) and exercise training sessions were performed 3 times per week (all EX groups). Rapamycin was delivered to mice via food at IACUC approved levels. Animals were treated a second time after 1 month of rest. During each treatment period, pre- and post-treatment performance assays were performed in eight mice (four males and four females of C57BL / 6J strain, aged 20–24 months) in each group (control (sham treatment), ultrasound (US), ultrasound + exercise (EXUS), exercise (EX) rapamycin (Rap), and rapamycin + ultrasound (RUS)).

[0091] 7A-7E show the effect of ultrasound treatment on the performance of aged mice. (FIG. 7A) Schematic of the treatment regimen. Mice (C57BL / 6J strain, 22-24 months old) were treated with LFU for 30 min every third day or ran on the treadmill for 20 min 12 times for one month. Mice were then rested for one month and treated or exercised for the second month. Each test group included 4 male and 4 female mice. Graphs of the results of the inverted cling test after (FIG. 7B) the first month and (FIG. 7C) the second month of treatment. (FIG. 7D) Results of the treadmill test after the first month and (FIG. 7E) results of the treadmill test after the second month of treatment. Groups were either untreated (sham), treated with LFU (US), treated with exercise (EX), treated with rapamycin (RAP), treated with LFU + exercise (EXUS), and treated with rapamycin + LFU (RUS). Results were plotted as mean ± SD. Statistical significance was determined using Student's t-test. P values ​​> 0.05 are represented by ns. Statistical significance was provided by p value. * p-value < 0.05, ** p-value < 0.001, and *** p-value<0.0001.

[0092] Example 7. Cellular rejuvenation of mouse tissues by LFU

[0093] Figures 8A-8E show that LFU reduces the fraction of senescent cells in kidney and pancreas. Groups of 10 (5 males and 5 females) 22-25 month old mice were treated with LFU every day (LFU_D1), every other day (LFU_D2), every third day (LFU_D3), and every day (LFU_1,3_D1) at 1.3x power. After 2-4 weeks of treatment and evaluation, mice were euthanized and kidneys and pancreas were collected for SA-β-galactosidase staining. (Figure 8A) SA-β-galactosidase stained kidney sections of sham-treated and LFU_D3 mice. β-galactosidase staining appears blue and is more intense in sections from sham-treated kidneys. (Figure 8B) Images of spleen sections stained with SA-β-galactosidase shown in color. Scale bars = 150 μm and 20 μm. (FIG. 8C) Quantification of β-galactosidase stained area in kidney sections. β-galactosidase staining was significantly reduced in mice treated with all LFUs. (FIG. 8D) Quantification of β-galactosidase staining in spleen sections. β-galactosidase staining was significantly reduced in mice treated with all LFUs. Results are plotted as mean ± SD. Statistical significance was determined using Student's t-test. P values ​​> 0.05 are represented by ns. Statistical significance is provided as p value. * p-value < 0.05, ** p-value < 0.001, and *** p value < 0.0001 (n = 10 mice).

[0094] Example 8. Ultrasound enhancement of wound healing

[0095] In tests on mouse skin wounds, it was found that ultrasound can enhance healing in young mice. An even greater relative enhancement of healing may occur in older animals via rejuvenation of senescent cells. One application is the treatment of diabetic foot ulcers, where older individuals generally have poor circulation due to diabetes and continue to have non-healing foot ulcers (see Figure 6).

[0096] Ultrasonic coupling. The primary objective of sound transmission was to create a relatively constant tensile stress in the membrane for a period on the order of one second, while limiting harmful heat generation by causing only acceptable friction at cell-cell adhesions. This is achieved with wavelengths larger than the cell diameter. The criterion of stationarity requires an acceleration force equivalent to the pressure resistance on the tissue, limiting excitation to less than 200 kHz. The wavelength of 200 kHz in tissue is approximately 7.5 mm, so frictional heating is generally not a limiting factor. Coupling of low frequency sound waves into tissue without excessive reflection requires a form of impedance matching. This is achieved using a contact material, which can be part of the transducer or immersed itself, that is of similar acoustic impedance to the tissue being treated. Unlike most ultrasound treatment methods, the methods herein are not generally, or at least not intentionally (unless otherwise specified in this text) aimed at depositing energy in the specimen. The attenuation in the low frequency range is very low, which may be 20% across the entire human body, so the energy delivered to any given volume of tissue is only a small fraction of the energy transmitted, which is also why the text includes complex details on how the US waves are ultimately absorbed after passing through the patient. This is fundamentally different from ultrasound in the MHz range, which deposits virtually all of its energy over several centimeters into the specimen. In most settings, especially in the ulcer and diabetic devices listed above, the transmitted acoustic waves are not actively coupled / destroyed after passing through the target tissue. This can be achieved by impedance-matched absorbers with scattering bubbles inside them. These special coupling and uncoupling requirements are inherent characteristics of some embodiments of the devices that may be used in the present invention.

[0097] Ultrasonic transducers. We used 16mm and 25mm diameter ring transducers made from PZT4 and PZT8 materials that can be effectively driven up to 100 volts per millimeter thickness (Beijing Ultrasonics 25x10x4 and 16x8x4 piezo ceramic rings). The surfaces of these piezo elements (or piezo stacks) are thick and pre-tensioned, allowing them to contract and expand, resulting in large amplitudes up to about 1.5 μm at low frequencies. An aluminum cone was attached to the ring to spread the field out to about 5 cm in diameter, with a resonant structure to maintain the amplitude and form a plane wave. Aluminum couples well to PZT materials, but is not matched to the water bath due to its very different acoustic impedance. We added a rubberized coating of epoxy and silicone to improve emission and suppress internal reflections.

[0098] To avoid the near field of the transducers, a 2λ (two wavelengths) deep 37°C water bath was used between the tops of the transducers to separate the samples. The samples themselves were floated on the water surface in standard multi-well plates (Thermo Scientific Nunclon Delta Surface 96 well plates). The samples were sealed to prevent water ingress under ultrasound irradiation. The well plates were suspended on PET foil with a rectangular opening at the bottom of the plate. Both the polymer and glass cover slips showed minimal beam intensity attenuation.

[0099] To eliminate standing wave patterns that result in spatial intensity variations, the base of the transducer was suspended and rested on an absorber foam. The effects of surface reflections were minimized by sweeping the frequency from 5% to 15%, resulting in approximately 80% uniformity at the center of the sound cone. This ultimately allowed control of the emitted force via the transducer drive voltage. Both a resistor network and a class A amplifier were used with interchangeable results.

[0100] Despite the shielded wiring, there was still significant radio noise radiated from the transducer, so the incubator cabinet was used as a shielding capsule. For outdoor use, a shielded cable was used to power the transducer.

[0101] Ultrasonic generator. All primary signals and sequences were created by an ArduinoDue microcontroller. This allowed for the duty cycle, sequence, and duration to be rapidly varied. The analog setup used to calibrate the entire system outputs waveforms through an Arduino digital-to-analog converter and amplified through a medium power amplifier (LM 675 from Texas Instruments) with a symmetrical ±24V supply. This drives a high frequency ferrite ring core (Richco Ferrite Ring Toroid Core, 31.5 x 19.3 x 8mm) isolated 1:5 transformer that provides up to 200V amplitude to the transducer. Any modern embedded controller with a DAC can provide the required output signal, so any controller and amplifier can be used.

[0102] At higher output levels, the generator was operated as a switching power supply derived from a single 24V supply. The 3.3V logic of the controller cut off two transistor switches (Infineon IRFP260MPBF 50A, 200V N-Channel MOSFETs) driving each input of the same transformer. Pull-up was provided by 6, 8, or 10 ohm power resistors with a dissipation ratio of 50W. The transformer inputs were clamped by varistors (EPCOS Varistor 8nF 20A 56V) in the switching design and to the power supply by diodes (1N5408) in the amplifier design. The tunable range was kept within the 50% bandwidth of the transducer design frequency, i.e. a 30kHz generator was used from 22.5kHz to approximately 37.5kHz. The controller outputs waveform samples at 1M samples / s. By storing 3 full waves in 100 samples it is possible to output an accurate 30 kHz, so 10000 times per second. Soft starting and stopping is provided by increasing or decreasing the amplitude over the 30 waves, i.e. the same time base provides a precise on-off cycle.

[0103] Calcium indicator dye-based assay. Cell samples were incubated with calcium indicator dye (4 mM Cal-520 AM, AAT Bioquest) for 1 h. Samples were then replenished with fresh culture medium and allowed to stabilize for 30 min before sonication.

[0104] Immunohistochemical staining and fluorescence microscopy. Samples were fixed with 4% paraformaldehyde (Thermofisher Scientific) solution for 10 min and permeabilized with 0.2% TritonX100 for 5 min. Normal goat serum (2%) was used as a blocking buffer, and samples were treated with serum for 1 h. Samples were then incubated with rabbit polyclonal anti-Piezo1 primary antibody (1:200, Novus Biologicals, catalogue no. NBP1-78446) at 4°C overnight, and then treated with Alexa Fluor-594 secondary antibody (Thermofisher Scientific). Hoechst dye (1:1000, Thermofisher Scientific) was used to stain nuclei.

[0105] For in vitro studies, fluorescent and brightfield images were acquired using a widefield Olympus live-EZ microscope equipped with a Photometrics CoolSNAP K4 camera and a W1 live-SR spinning disk microscope equipped with a Photometrics Prime 95B sCMOS camera. For in vivo tumor imaging, a widefield Zeiss stereo microscope was used.

[0106] Apoptosis and necrosis assays. To identify cell apoptosis, Annexin V-Alexa Fluor 488 or Annexin V-Alexa Fluor 594 conjugates (Thermofisher Scientific) were used according to the manufacturer's protocol. To confirm cell necrosis, propidium iodide from a live / dead cell dual staining kit (Sigma Aldrich) was used according to the manufacturer's protocol. Assays were performed at least 12 hours after sonication.

[0107] Assay for viable cells. Cell viability was confirmed using calcein AM (Sigma Aldrich) dye according to the manufacturer's protocol. Cells were incubated with the dye for 25 min each day before imaging.

[0108] Cell lines and cell culture. Human foreskin fibroblasts (HFF) were purchased from ATCC. Vero cells, derived from African monkey kidneys, were donated by M. Garcia-Blanco lab. All these cell lines were cultured as per the manufacturer's protocol. Vero and HFF cells were in growth medium containing DMEM and 10% FBS. Cells seeded at 20–40% confluence were incubated at 37°C and 5% CO. 2 The cells were maintained in an incubator.

[0109] Induction and quantification of senescence. Vero cells were cultured in 200 μM HO. 2 O 2 The cells were treated with various stressors, including 10 μM sodium butyrate (SB), 4 μM sodium butyrate (SB), and 25 μM bleomycin sulfate (BS), and incubated for 36–48 h. After washing with PBS and then replacing the growth medium with fresh medium, the cells were incubated for 4 days to confirm the arrest of senescent cell growth. Human foreskin fibroblasts (HFFs) were passaged stepwise up to p15, because the replication of these cells is significantly reduced at p15-17. We used four criteria to determine whether the cells were senescent: (1) cell cycle arrest by determining the growth rate, (2) increase in the spread area of ​​the cells, (3) development of a senescence-associated secretory phenotype (SASP) in the culture medium, and (4) β-gal staining. We obtained images of the cells using an Evos microscope at 10x magnification after treatment and 48 h after treatment. To measure growth due to increasing cell numbers, 15 random images were acquired and then the average number of cells was determined, which was then divided by the area of ​​one frame to obtain the cell density (cells / cm). 2 ) was obtained. This seeding density was then multiplied by the total area of ​​the culture dish or well to obtain the total number of cells after US treatment and after 48 hours if incubated. The total number of cells at 48 hours was divided by the total number of cells immediately after treatment to determine the growth rate. A ratio of 1 indicates no growth.

[0110] Senescence was detected by β-gal senescence staining kit as per the manufacturer's protocol. Briefly, subconfluent senescent cells were stained with SA-β-gal staining solution and incubated overnight at 37°C. Cells stained with β-gal appeared blue and were senescent cells. The percentage of β-gal positive cells was determined by counting the number of blue cells and dividing by the total number of cells. The spreading area of ​​cells was determined by obtaining an image of the cells with a 10x objective lens using an Evos microscope. We then calculated the spreading area by manually encircling the cell periphery of each cell using ImageJ software. We used a minimum of 150 cells for analysis. To determine the activity of SASP, we cultured senescent cells for 3-4 days and then collected the supernatant from each culture dish. This supernatant was used to culture normal cells. The development of a senescent phenotype by normal cells in the supernatant medium confirmed that senescent cells were secreting SASP.

[0111] Sonication of cells. Prior to sonication, plates containing senescent Vero cells or late passage HFF cells were wrapped in parafilm to avoid contamination and water flowing into the plate. Samples were placed on a plastic mesh mounted on a water tank equipped with an ultrasonic transducer. The water in the tank was degassed and heated to a temperature of 35°C. The distance between the sample and the transducer was approximately 9-10 cm. We also ensured that there were no air bubbles or air-water interfaces between the water and the sample. The output of the transducer was measured by a hydrophone at the plate. Cells were treated with pressure pulses of 3.5-4.0 pa using ultrasound at a frequency of 32.249 kHz for 30 minutes. Cells were treated with cycles of 1.5 seconds on and 1.5 seconds off. After sonication, the cell plates were returned to the incubator for 48 hours to determine the growth of senescent cells.

[0112] Reversal of senescence. First, we induced senescence in Vero cells using sodium butyrate, then we confirmed senescence using growth arrest and Betagal staining methods. We treated senescent cells using ultrasound with optimized parameters (33 kHz frequency and 3.5-4 pa). We incubated the cells for 48 hours, measured cell growth and morphology, and then trypsinized. We designated this passage as P0. Then, the cells were trypsinized, reseeded, and incubated for 48 hours at passage P1. This process was repeated until passage P3. We evaluated the cells in terms of fold change in proliferation, morphology, β-gal, and EDU staining to confirm the senescent cell population. Senescent cells without ultrasound treatment were used as control cells. Usually, until passage P3, the senescent cells exhibited the phenotype of normal proliferating cells.

[0113] HFF cells at passages 15-24 were treated with ultrasound at optimized frequencies and powers. Cell proliferation was determined by counting the number of cells at the time of seeding and 48 hours after US treatment. HFF cells treated with ultrasound showed a higher fold change in growth than untreated HFF cells. For P24 HFF cells, they were treated with ultrasound, incubated for 96 hours, trypsinized, reseeded, and incubated for 48 hours. After 48 hours of incubation, proliferation and morphology were measured. HFF cells treated with US at P24 were reduced in size compared to untreated P24 HFF cells, and they also showed significantly more proliferation.

[0114] Ultrasound treatment of aged mice. Aged mice (21-24 months old) were treated in a large 4 L glass beaker with one plastic cylinder with a height of 13 cm and a diameter of 152 cm. A metal mesh that supported the mouse and allowed the mouse's four limbs and body to rest in the water was placed on top of the cylinder. Degassed 32-35°C warm water was poured into the beaker. The water level was kept 1 inch above the metal mesh so that half of the mouse's body remained underwater. After placing the mouse in the water, intermittent ultrasound at 32.249 kHz and 3.5-4 Pa was applied to the mouse for 1.5 seconds on and 1.5 seconds off for 30 minutes. The animals in the ultrasound group were treated for 1 month with an interval of 72-96 hours (10 treatments). During the ultrasound treatment, the activity of the mice and their adaptation to the system were observed. After the treatment, the animals were placed in separate cages with tissue paper to dry the animals and then returned them to their home cages. Control mice were placed in the same water bath for 30 minutes without ultrasound. The ultrasound treatment procedure involved placing the animals in direct contact with the water. The reason for placing the animals in water is that ultrasound is significantly attenuated at the air-water interface.

[0115] Physical evaluation of mice. For the evaluation of the effect of ultrasound (US) treatment on the physical performance of mice, we used six groups of aged mice: 1. sham treatment, 2. ultrasound treatment, 3. exercise, 4. rapamycin, 5. exercise + ultrasound, and 6. ultrasound treatment + rapamycin. Each group consisted of 4 males and 4 females. For animals treated with rapamycin, C57BL / 6J mice were given encapsulated rapamycin and monitored daily for one month. Animals in the ultrasound group were treated every 72-96 hours for one month. Animals in the exercise group were trained on a treadmill for 25 minutes three times per week in each exercise training session. Before the start of the experiment, the physical function and health of the mice were evaluated, which was called pre-evaluation. After one month of ultrasound treatment and exercise sessions, the physical performance and health of the animals were evaluated again, which was called post-evaluation. Physical performance was determined by functional assessment tests including the Grip Test, Rotarod, Treadmill, and Inverted Cling Test.

[0116] Treadmill. Mice were tested for maximum force output / maximum walking speed and endurance (until fatigue) by running on a treadmill. The measurement of output was the running duration. During the training session, mice were habituated to the apparatus and initially ran at a constant speed, which was then gradually increased by one unit every 20 seconds. Mice were allowed to rest for 10 minutes between trials. Finally, the trial was terminated with three electric shocks of 0.4 mA, and the animals were given three trials. During the test session, the speed was increased and the mice were allowed to run as far as they could before receiving three shocks.

[0117] Inverted hold: This grip test was useful for quantifying muscle strength and endurance by measuring how long a mouse could hold on to a grid while upside down. Each animal was tested twice, with a 10 minute rest between trials. A minimum of 10 seconds of holding was required for validation of the test to eliminate slippage. Three trials were performed in the first test, followed by two trials after a 10 minute gap.

[0118] Exercise training sessions. Animals in the exercise and exercise + ultrasound groups were exercised on a treadmill for 25 min every 48 h, three times a week for one month. All exercise sessions were preceded by a 10 min warm-up at 6 cm / s, followed by 10 min training at 8 cm / s and a 6 cm / s cool-down. Training began progressively at 8 cm / s in week 1 and increased to 11 cm / s in week 4. Mice were encouraged to run on the treadmill with a light electric shock when they stopped running.

[0119] There were 12 exercise sessions and 10 ultrasound treatments over the course of the one-month study.

[0120] Mitochondrial morphology. Cells treated with ultrasound were incubated with Mitotracker (Invitrogen) at 100 Um for 30 min at 37°C. Images were then acquired with a confocal microscope with 15 random fields per sample for quantification. AR and form factor were determined using the formula major axis / minor axis and circumference2 / (4π×surface area)1.

[0121] Immunofluorescence staining. Cells were seeded on 27 mm glass-bottom dishes (Ibidi) after sonication, and the cells were washed twice with PBS. They were then fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 for 5 min. The cells were washed three times with PBS and incubated with 3% blocking buffer for 1 h. Anti-LAMP primary antibody (1:400) was incubated with the samples overnight at 4°C, followed by incubation with Alexa flour 488 secondary antibody (1:1000) for 4 h at room temperature. Images were obtained by confocal microscopy.

[0122] Mitochondrial ROS. MitoSOX (Invitrogen) red was used to measure mitochondrial reactive oxygen species production. Briefly, 5 μl of MitoSOX was added to growth medium for 10 min at 37°C. Fluorescent images were obtained with a confocal microscope (Olympus). ROS levels were determined by intensity measurements.

[0123] Viable and dead cell assay. Viable cells were detected using Calcein AM (Sigma Aldrich) according to the manufacturer's instructions. Briefly, adherent cells were treated with Calcein AM 2000:1 in Opti-MEM medium and incubated for 30 min. Apoptotic / dead cells were identified using Annexin V-FITC / Propidium Iodide (PI) (Sigma Aldrich) according to the manufacturer's instructions. Live / dead assays were performed immediately after ultrasound exposure and after 24 h of treatment.

[0124] Calcium release assay. Calcium release was measured using calcium dye (4 mM Cal-520 AM, AAT Bioquest) according to the manufacturer's protocol. Semi-confluent cells were incubated in calcium dye for 30 min before sonication.

[0125] Statistical analysis. All results are presented as mean ± sd and paired two-tailed t-tests were used for two groups. Graphs were generated and statistical analysis was performed using GraphPad Prism 8.4.3. As shown in the figures, * P value < 0.05, ** P value < 0.002, *** A P value of <0.001 and non-significant (ns) P value >0.05 were used.

[0126] It is contemplated that all embodiments discussed herein can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Further, a composition of the invention can be used to achieve a method of the invention.

[0127] It will be understood that the specific embodiments described herein are shown by way of example and not by way of limitation of the invention. The principal features of the invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the invention and are covered by the claims.

[0128] All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0129] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification can mean "one" as well as "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and / or," unless expressly stated to represent only alternatives or the alternatives are not mutually exclusive, although the disclosure supports a definition that represents alternatives and "and / or" only. Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of equipment error and the method used to determine the value or variation that exists among test subjects.

[0130] As used in the specification and claims, the terms "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In any of the embodiments of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of." As used herein, "consisting essentially of" requires the recited integers or steps and integers or steps that do not materially affect the features or functions of the claimed invention. As used herein, the term "consisting of" is used to denote only the presence of a recited integer (e.g., a property, element, feature, quality, method / process step, or limitation) or group of integers (e.g., a plurality of properties, elements, features, properties, method / process steps, or limitations).

[0131] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB, if the order is important in the particular context. Continuing with this example, combinations including one or more repeats of an item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, are expressly included. Those skilled in the art will generally understand that no limitation is made with respect to the number of items or terms in any combination, unless otherwise clear from the context.

[0132] As used herein, without limitation, approximation terms such as "about", "substantial" or "substantially", when so modified, are not necessarily understood to be absolute or complete, but rather represent a state that is considered close enough to those skilled in the art to reliably specify the state that exists. The degree to which the description may vary depends on how large a change is made, but still allows those skilled in the art to recognize that the modified property has the required characteristics and attributes of the unmodified property. Generally, subject to the preceding discussion, numerical values ​​herein modified by approximation terms such as "about" may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12, or 15%.

[0133] Further, the section headings herein are provided for consistency with the teachings under 37 CFR 1.77 or to provide organizational cues. These headings do not limit or characterize the invention(s) set forth in any claims that may arise from this disclosure. Specifically and by way of example, a heading may refer to the "Field of the Invention," but such claims should not be limited by the language under this heading to describe the so-called technical field. Furthermore, the description of a technology in the "Background of the Invention" section should not be construed as an admission that the technology is prior art to any invention in this disclosure. The "Summary" is not to be considered a characterization of the invention(s) set forth in the claims that will be issued. Furthermore, any reference to "invention" in the singular in this disclosure should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth by the limitations of multiple claims that will be issued from this disclosure, and such claims thus define the invention(s) and their equivalents that are protected thereby. In all instances, such claims are to be considered as unique advantages in light of this disclosure, but should not be limited by the headings set forth herein.

[0134] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention are described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the compositions and / or methods, and in the steps or sequence of the methods described herein, without departing from the concept, spirit, and scope of the invention. Such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention, as defined by the appended claims.

[0135] In order to assist the Patent Office, and any reader of any patent that may issue on this application, in interpreting the claims appended hereto, Applicants wish to note that none of the appended claims, or equivalents, are intended to invoke paragraph 6 of 35 U.S.C. 112, paragraph (f), as it exists on the filing date, unless the words "mean for" or "step for" are clearly used in a particular claim.

[0136] In each claim, each dependent claim may depend on both the independent claim and each preceding dependent claim with respect to any claim, provided that priority provides appropriate antecedent for a term or element of the claim.

[0137] References 1.Gorgoulis, V. et al. Cellular Senescence: Defining a Path Forward. Cell 179, 813-827 (2019). 2.Krimpenfort, P. & Berns, A. Rejuvenation by Therapeutic Elimination of Senescent Cells. Cell 169, 3-5 (2017). 3.Neves, J., Sousa-Victor, P. & Jasper, H. Rejuvenating Strategies for Stem Cell-based Therapies in Aging. Cell Stem Cell 20, 161-175 (2017). 4.Blood factors transfer beneficial effects of exercise on neurogenesis and cognition to the aged brain | Science. https: / / science.sciencemag.org / content / 369 / 6500 / 167. 5.Hubackova, S. et al. Selective elimination of senescent cells by mitochondrial targeting is regulated by ANT2. Cell Death Differ. 26, 276-290 (2019). 6.Baar, M. P. et al. Targeted Apoptosis of Senescent Cells Restores Tissue Homeostasis in Response to Chemotoxicity and Aging. Cell 169, 132-147.e16 (2017). 7.Kirkland, J. L. & Tchkonia, T. Cellular Senescence: A Translational Perspective. EBioMedicine 21, 21-28 (2017). 8.Cai, Y. et al. Elimination of senescent cells by β-galactosidase-targeted prodrug attenuates inflammation and restores physical function in aged mice. Cell Res. (2020) doi:10.1038 / s41422-020-0314-9. 9.Libertini, G., Ferrara, N., Rengo, G. & Corbi, G. Elimination of Senescent Cells: Prospects According to the Subtelomere-Telomere Theory. Biochem. Mosc. 83, 1477-1488 (2018).

Claims

1. A low-frequency ultrasound system for use in reversing aging by applying repetitive low-frequency ultrasound treatment to at least one viable cell, wherein the repetitive low-frequency applies a periodic force having a duration, magnitude, and frequency sufficient to deform the at least one viable cell so as to induce a mechanically induced aging reversal process without causing mechanical or thermal damage to the at least one viable cell.

2. 10. The low frequency ultrasound system for use according to claim 1, wherein the wavelength of the low frequency ultrasound treatment is equal to or greater than the average cell diameter.

3. A low-frequency ultrasound system for use as described in claim 1, wherein reversing aging includes at least one of reducing cell size, increasing secretion of growth factors, or preventing mitochondrial fusion.

4. 2. The low frequency ultrasound system for use according to claim 1, wherein the repetitive low frequency ultrasound treatment is at 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 750, 800, or 900 kHz.

5. The repetitive low frequency ultrasound treatment is 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1,000, 5,000, 10,000, or <500 mW / cm ２ 2. A low frequency ultrasound system for use according to claim 1, wherein:

6. 10. The low frequency ultrasound system for use according to claim 1, wherein at least a portion of the applications of the repetitive low frequency ultrasound treatment are applied simultaneously with an active agent that reduces cellular senescence.

7. 7. The low frequency ultrasound system for use according to claim 6, wherein said active agent is the supernatant of at least one viable cell treated with said repetitive low frequency ultrasound treatment.

8. The repetitive low frequency ultrasound treatment comprises: one or more ultrasonic transducers configured to produce a sequence of waves of programmed cycles; one or more ultrasonic transducers attached to the robotic arm; one or more ultrasonic transducers, wherein the robotic arm is controlled to position the one or more ultrasonic transducers in a desired position or orientation; one or more ultrasonic transducers, which may be phased array ultrasonic transducers or which are sealed and water-resistant; and delivered from at least one of the frequency, magnitude, and duration of the periodic force are determined based at least in part on the type of target cell or based on the output of a feedback sensor near the treatment zone; or The wavelength of the wave is on the order of the size of the organ, and the amplitude is on the order of a single cell. A low frequency ultrasound system for use according to claim 1.

9. 10. The low frequency ultrasound system for use according to claim 1, wherein the repetitive low frequency ultrasound treatment is delivered to at least a localized region of a patient's body.

10. 10. The low frequency ultrasound system for use according to claim 1, wherein the at least one viable cell is a plurality of viable cells, and the repetitive low frequency ultrasound treatment extends the replicative lifespan of the at least one viable cell.

11. 10. The low frequency ultrasound system for use according to claim 1, wherein the at least one viable cell is a plurality of viable cells, and the repetitive low frequency ultrasound treatment reverts the plurality of viable cells to a younger phenotype.

12. A low-frequency ultrasound system for use as described in claim 1, wherein the repetitive low-frequency ultrasound treatment does not induce spontaneous cell death in normal cells.

13. A low-frequency ultrasound system for use as described in claim 1, wherein the shape of at least one viable cell may be deformed by about 1% to about 5%, about 3% to about 8%, or about 5% to about 10%.

14. A low frequency ultrasound system for use as described in claim 1, wherein the repetitive low frequency ultrasound treatment is applied for approximately 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 24 hours, 48 ​​hours, or more.

15. A low frequency ultrasound system for use as described in claim 1, wherein the repetitive low frequency ultrasound treatment has a frequency in the range of approximately 30 kHz to 250 kHz.

16. A low frequency ultrasound system for use as described in claim 1, wherein the repetitive low frequency ultrasound treatment is non-focused.

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