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"I am Exposing the Whole Damn Thing!" (MIND BLOWING!!!!) | Randall Carlson

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https://www.sciencedirect.com/topics/chemistry/vibrational-frequency

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Sound waves power new advances in drug delivery and smart materials
https://www.sciencedaily.com/releases/2020/11/201124101029.htm

Sound waves have been part of science and medicine for decades, but the technologies have always relied on low frequencies. Now researchers have revealed how high frequency sound waves could revolutionize the field of ultrasound-driven chemistry.

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Science Talk - The quiet revolution: how high-frequency sound waves are being used to treat cancer pain - The Institute of Cancer Research, London

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When you hear the word ultrasound what springs to mind? Perhaps pregnancy scans or bats flying in the night sky?

Ultrasound is the name used to describe sound waves that have frequencies too high to be detected by the human ear. They are used in nature by bats and porpoises for navigation, and by humans in a whole host of industrial and medical applications, from measuring distances, to accelerating chemical processes, to health diagnostics.

The beauty of ultrasound is its ability to go unnoticed and not leave a trace - except to those capable of detecting the high-frequency waves.

So it might come as a surprise to know that researchers and clinicians at The Institute of Cancer Research and The Royal Marsden are using ultrasound, focused to be 10,000 times more powerful than that used in pregnancy scans, to destroy nerve tissue to treat cancer pain.

The technique, known as high-intensity focused ultrasound (HIFU), concentrates high-power sound waves to create heat in the same way that a magnifying glass can concentrate sunlight to burn paper. Ultrasound waves can be focused so intensely that they can destroy tissue when precisely targeted within the body.

We recently invited a small group of journalists to come and see the HIFU facility and to meet experts working in the area, resulting in media coverage which included a piece in the Observer. Researchers and clinicians at the ICR and Royal Marsden have been central to a growing movement to explore the potential of ultrasound as a treatment. In 2013 the ICR and the Royal Marsden became a Focused Ultrasound Foundation centre of excellence, and last year we embarked on a clinical trial to use focussed ultrasound to burn away nerve endings in patients with advanced cancer with bone metastases.

The trial is part of a wider initiative between the ICR, The Royal Marsden, the Focused Ultrasound Foundation and Philips, the developer of the HIFU system. This collaboration is creating a state-of-the-art resource for clinicians and scientists to establish global standards for focused ultrasound and accelerate development of the technology to treat cancers.

Professor Gail ter Haar, Professor of Therapeutic Ultrasound at the ICR, has been instrumental in pioneering ultrasound as a potential cancer treatment. She told me: "We see instant cell death if we achieve a temperature of 50°C in tissue, and hold it for one second using HIFU, and we can deliver this very accurately, to within a six cell margin.

"We use state of the art MRI scanning to guide delivery of focused ultrasound during the treatment. Not only does it allow us to monitor how hot the tissue is getting, it also provides very good contrast so we can precisely target the correct area."

Professor Nandita deSouza, Professor of Translational Imaging at the ICR and Consultant Radiologist at The Royal Marsden, gave us an update on the trial: "We have now used HIFU to treat a number of patients with advanced cancer. The patients we are treating are in a lot of pain - tumours that have spread to their bones are pushing against nerve endings. Other treatments have failed, and there are no options left to them. HIFU has given us a way to reduce the pain these patients are experiencing. Remarkably, as tissue dies instantly, some of our patients have seen an immediate effect in terms of pain reduction."

Though HIFU as a cancer treatment is at an early stage of development, it is hoped in the long term that the technique could be used to destroy tumours. The areas surrounding the treatment site are unlikely to be affected; ultrasound does not harm cells when it is not generating heat. This means it may hold promise for patients who are unable to have surgery or who have already reached a maximum dose of radiotherapy.

"We are really excited about the potential of this technique. We can see how it could benefit patients, but we are still some way from offering this to patients as a cancer treatment," said Prof deSouza. "However, if the results from our trial continue to be positive, we would hope to run further trials directly targeting tumours with HIFU for patients who have no other treatment options.

"This treatment is very patient-friendly; it involves no invasive procedures as it can be delivered from outside of the body.

"In the long term, we hope that we could develop HIFU as a treatment for cancer for doctors to have in their toolkit alongside established treatments such as radiotherapy, surgery, chemotherapy and targeted treatments."

So the day that we see ultrasound used as a treatment as well as a diagnostic may be approaching. Watch this space for regular updates.

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Science Talk - The quiet revolution: how high-frequency sound waves are being used to treat cancer pain - The Institute of Cancer Research, London

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When you hear the word ultrasound what springs to mind? Perhaps pregnancy scans or bats flying in the night sky? Ultrasound is the name used to describe sound waves that have frequencies too high to be detected by the human ear.

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https://onlinelibrary.wiley.com/doi/10.1002/advs.202001983

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Vibration detection: its function and recent advances in medical applications - PubMed

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Huge Medical Advancements In Vibrational Therapy

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Huge Medical Advancements In Vibrational Therapy

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Affiliations

¹ Molecular Neurobiology, Groningen Institute for Evolutionary Life Sciences (GELIFES), University of Groningen, Groningen, The Netherlands.

² Research Center for Molecular Exercise Science, University of Physical Education, Budapest, Hungary.

³ Department of Human Movement Sciences, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

Free PMC article

Free PMC article

Affiliations

¹ Molecular Neurobiology, Groningen Institute for Evolutionary Life Sciences (GELIFES), University of Groningen, Groningen, The Netherlands.

² Research Center for Molecular Exercise Science, University of Physical Education, Budapest, Hungary.

³ Department of Human Movement Sciences, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

Abstract

Vibrations are all around us. We can detect vibrations with sensitive skin mechanoreceptors, but our conscious awareness of the presence of vibrations is often limited. Nevertheless, vibrations play a role in our everyday life. Here, we briefly describe the function of vibration detection and how it can be used for medical applications by way of whole body vibration. Strong vibrations can be harmful, but milder vibrations can be beneficial, although to what extent and how large the clinical relevance is are still controversial. Whole body vibration can be applied via a vibrating platform, used in both animal and human research. Recent findings make clear that the mode of action is twofold: next to the rather well-known exercise (muscle) component, it also has a sensory (skin) component. Notably, the sensory (skin) component stimulating the brain has potential for several purposes including improvements in brain-related disorders. Combining these two components by selecting the optimal settings in whole body vibration has clear potential for medical applications. To realize this, the field needs more standardized and personalized protocols. It should tackle what could be considered the "Big Five" variables of whole body vibration designs: vibration amplitude, vibration frequency, method of application, session duration/frequency, and total intervention duration. Unraveling the underlying mechanisms by translational research can help to determine the optimal settings. Many systematic reviews on whole body vibration end with the conclusion that the findings are promising yet inconclusive. This is mainly because of the large variation in the "Big Five" settings between studies and incomplete reporting of methodological details hindering reproducibility. We are of the opinion that when (part of) these optimal settings are being realized, a much better estimate can be given about the true potential of whole body vibration as a medical application.

Keywords: Meissner corpuscles; Pacinian corpuscles; Whole body vibration; brain; cognition; exercise; somatosensory; translational research.

Conflict of interest statement

No competing interests were disclosed.No competing interests were disclosed.No competing interests were disclosed.

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Mortimer B: Biotremology: Do physical constraints limit the propagation of vibrational information? Anim Behav. 2017;130:165-74. 10.1016/j.anbehav.2017.06.015 - DOI

Endler JA: The emerging field of tremology.In: Crofort RB, Gogala M, Hill PSM, Wessel A, editors). Studying vibrational communication Heidelberg: Springer;2014;vii-x.

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Grant support

The author(s) declared that no grants were involved in supporting this work.

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# data[].attributes.attachment_link.title
Vibration detection: its function and recent advances in medical applications - PubMed

# data[].attributes.attachment_link.description
Vibrations are all around us. We can detect vibrations with sensitive skin mechanoreceptors, but our conscious awareness of the presence of vibrations is often limited. Nevertheless, vibrations play a role in our everyday life. Here, we briefly describe the function of vibration detection and how it ...

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Sound waves power new advances in drug delivery and smart materials

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Sound waves power new advances in drug delivery and smart materials

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How high-frequency sound waves can be used to build new materials, make smart nanoparticles and deliver drugs for painless, needle-free vaccinations.

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Possible Mechanisms for the Effects of Sound Vibration on Human Health

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by 1,* and 1,2

Music has the power to affect our health and wellbeing. This belief and related cultural practice has been evident throughout history [ 1, 2], but the clinical profession of music therapy is only some 75 years old. As a field of practice music therapy has been described as an art and a science and regards to science its research foundation has drawn on psychoanalytic, humanistic, and behavioral traditions [ 3]. As an emergent practice within the healthcare context, the focus of music therapy (a therapeutic relationship employing music as a means) and music medicine (the music or sound itself as the therapeutic) research has been primarily evidence-based. Mechanisms of effect have received little attention until recently. To develop clinical and scientific legitimacy in general healthcare and expand its use as standard of care, research on the use of music must show both that it works (evidence-based research) and why it works (mechanism-based research).

Part of the problem of creating a scientific foundation for music effects in healthcare is that music is a very complex phenomenon; to control its parameters scientifically is practically impossible. That is, if music is approached as "music"-a cognitively constructed product of a culturally situated acoustic phenomena-the variables that can affect a response are practically limitless. Efforts to provide more detailed reporting of the music used in research are a start [ 4]. However, another approach is to define the potential level of the response mechanism.

1.1. Basic Concepts

1.1.1. Mechanism

Focusing on the mechanism itself can be highly complex. Craver and Bechtel [ 5] describe four dimensions of mechanisms: (1) a phenomenal dimension-a mechanism does things. For music the human ear offers a basic mechanism illustration. The ear translates compression and decompression of air molecules into the conscious perception of sound. (2) A componential dimension-a mechanism has components. In a simplified description the ear has components including the ear drum, the malleus, incus, stapes, and cochlea that transmit the vibration to the cilia which translate the physical vibration to an electrical signal for the auditory nerve. (3) A causal dimension-components interact to make the phenomenon happen. The components of the ear turn the air compressions into electrical signals transmitted through the auditory nerve to the internal perceptual processing system (a complex mechanism in itself). (4) An organizational dimension-components are organized in space and time. There is a particular organization of the components of the ear and how they interact that causes vibration to be perceived as sound.

The basic question of what are the mechanisms of how people enjoy music illustrates a very important aspect of the challenge of studying mechanisms-the ear is essential in the perception of music but it is only a start to the understanding of the cascade of mechanisms or causes of music enjoyment. That understanding would involve further definition of the auditory nuclei in the brainstem, the role and function of the thalamus, the means of transmission to the auditory cortex, the role of memory in the perceptual process, the interaction of expectation with neurotransmitters like dopamine and their role in the perceived response, and so on. Similarly, the question of how does low frequency sound stimulates blood circulation leads to an examination of endothelial cell response resulting in nitric oxide release and its effect on blood flow and so on.

The crucial point here, and an important caveat for this paper, is that research into the mechanisms of the effect of music on health is nascent and in most cases focused on the most obvious components of what can be presumed to be a complex system [ 6, 7]. This paper will, therefore, attempt to point in the direction of potential mechanisms that appear to be emerging for further study rather than serve as a detailed explication of a set of defined mechanisms.

1.1.2. Types of Stimulus and Response

A starting point to study mechanisms is to try to categorize by type of stimulus and type of response. One of the difficult pivots in the thinking of many health researchers is to differentiate between "music" and "sound vibration" as the stimulus. The bulk of research by cognitive neuroscientists focuses on "music" and often with little control of the parameters of that music since the assumption is that the effect stems only from the cognitive processing of that music [ 8, 9].

Within the broad category of "music" as stimulus, the response focus can be on the general effects like arousal, mood, emotion, relaxation, stress, etc., and with the assumption that these responses result from enjoyment or aversion reactions, memory associations, or some inherent neurological process [ 8]. All of these responses can be attributed to learned schemas, processes, and associations developed through socially mediated cultural immersion from the very earliest days of exposure to music [ 10].

A second category of mechanisms within "music" is to look at the effect of music cognition on the activation of neural circuits and specific neural functions. This has been most clearly defined in relation to rehabilitation [ 11, 12].

1.1.3. Vibratory Rhythmicity

This paper will not focus on music as "music"; it will focus exclusively on the rhythmicity of music and sound as vibration. In many cases the focus is on the effect of single frequencies although these frequencies may be imbedded in music and heard by the recipient simply as music.

1.2. Vibratory Applications to Health

1.2.1. Historic Predicates

It is common to encounter studies of the effect of vibration and low frequency sound in industrial-oriented research. These studies typically focus on the potentially negative effects of specific work environments, use of particular equipment, and primarily exposure to low frequency vibration and noise [ 17, 18]. However, there is also a long history of interest in the positive effects of vibration. Carriage rides on rough cobblestones came to be associated with positive health effects and mechanical "chairs" emulating these rides were created. In the 18th century the Abbéde St. Pierre created the tré moussoir or fauteuil de poste as a vibrating chair to help patients with melancholia, liver disease, and other conditions that seemed to respond to carriage rides [ 19]. The 19th century saw considerable interest in vibration and the development of vibrating tools including the chair created by Dr. Jégu after seeing Parkinson's disease (PD) symptoms calmed by a carriage ride [ 19, 20]. Dr. Jean-Martin Charcot, the most noted neurologist of that century, began studying PD with the chair but acknowledged an earlier physician, M. Vigoroux, who in 1878 used a sounding box with a very large attached tuning fork played with a bow to successfully treat patients with hemianesthesia and locomotor ataxia [ 21]. Charcot also described treatment of neuraglia and migraines by Dr. Boudet of Paris with tuning forks mounted on small boards. Gilles de la Tourette, Charcot's assistant, applied the idea of vibration therapy to the brain [ 19, 20, 21] with a metal helmet he devised with a motor on top causing the helmet to vibrate at 10Hz. It was found to have a positive effect on insomnia, migraines, and depression and other vague conditions [ 21].

1.2.2. Current Therapeutic Application Concepts for Vibration

More in keeping with the early uses of tuning forks for sensory stimulation is the practice of low frequency sound therapy (and most closely related to music therapy) that has developed in the past 50 years and is now commonly known as vibroacoustic therapy (VAT). WBV typically uses frequencies below 30 Hz while VAT uses 30-120 Hz. Olav Skille in Norway and Petri Lehikoinen in Finland were the leaders in this use of sinusoidal sound to stimulate the body for therapeutic purposes. Skille placed particular emphasis on single pitches at 40, 52, 68, and 86 Hz modulated with a steady rise and fall of amplitude at a rate of about 6.8 s from peak to peak. A noteworthy application of this method in research was done by Wigram [ 25, 26]. Instead of single frequencies, Lehikoinen used constant frequency scanning with the intent to treat muscles at their particular resonance frequency, slow power pulsation to prevent muscle contraction, and at times directional movement of the sound [ 27]. Lehikoinen developed the NextWave chair system that was Federal Drug Administration (FDA) and British Standards Institution (BSI) approved in 1996 for three claims related to physioacoustic therapy: increased blood and lymphatic circulation, decreased pain and stress, and increased muscle relaxation and mobility [ 28]. Numerous devices that include chairs, chair backs, beds, mats, pillows, backpacks, and smaller hand-held units have been developed since then.

1.3. Definitions, Clarifications, and Terminology

1.3.1. Source of Pulsed Stimulation: Sound Waves or Mechanical Compression

To review the literature related to vibration, one of the fundamental questions requiring clarification is whether the stimulation of the body with sound waves is different in some way from mechanical vibration. Since people hear sound waves and feel mechanical vibrations, an easy conclusion is that the two are categorically different. However, sound in essence is mechanical vibration that transmits through a medium [ 29]. In the medium of air, the sound actuator creates a vibration that results in regular compressions and decompressions of air molecules that travel to the receiving surface on the body such as the ear's tympanic membrane or the mechanoreceptors in the skin. In water a rapidly oscillating membrane would create compressions and decompressions of water molecules. Once in contact with the body, the compression and decompression of the surface of the body is transmitted through bone and tissue and may be sensed by a set of mechanoreceptors or by our auditory system.

At a cellular or molecular level in the body there is probably no difference between activation by air molecules applying regular sine wave pressure on the body, by a surface applying oscillatory pressure stemming from a rotating motor shaft, or by the body itself moving against gravity on an oscillating platform. Another way to understand this can be in comparing the application of sound to produce vibration and the application of vibration to produce sound. Sound, which propagates through a material can vibrate the material and be physically felt like a massage, such as in a vibroacoustic device [ 30]. Mechanical vibration can also produce sound. Bone conduction headphones are commercially available and are built to be positioned along the skull. The mechanical vibration of the bones of the skull propagate to the inner ear and are perceived as sound [ 31]. Therefore, the interchange of sound and mechanical vibration demonstrate that they are in essence the same thing.

1.3.2. Vocabulary and Inclusion

Clarification is needed about the vocabulary used in this field. We have already explained VAT and WBV. Another term used is rhythmic sensory stimulation (RSS) and is inclusive of multiple types of pulsed (rhythmic) stimulation. RSS includes whole body rhythmic movement, vibrotactile stimulation of all or part of the body, auditory pulses delivered as individual sound units (like hits on a drum, plucks of a string, interaction "waves" resulting from binaural detunement, or isochronous amplitude modulated sound trains) or as molecular compressions that create continuous sound (research usually focusing on low frequency sound 20-130 Hz), and visual light flashes or flicker. Pulsed ultrasound can also be regarded as a type of vibrational mechanical stimulation and is typically applied in 2 ms bursts but with varying ratios of sound to silence from 1:1 (250 Hz) to 1:20 (45 Hz).

When considering the use of rhythmic pulses to stimulate to the body, one question is whether electric stimulation and pulsed ultrasound are comparable to mechanical vibration. Bartel et al. [ 32] drew parallels between pulsation frequencies used in electric stimulation and pulsed ultrasound with frequencies for VAT. Many years ago Charcot postulated that the vibration stimulation was not unlike electric stimulation and showed similar results [ 21]. In this paper the review of specific mechanisms activated by vibration will be restricted to studies using various sonic and mechanical vibratory means from general body vibration to focused points of vibration delivered with pencil-like probes. However, a future more extensive review of the mechanisms activated by pulsed stimulation might include electrical and pulsed ultrasound stimulation as well.

A final question is whether whole body vibration (WBV) is fundamentally different from vibroacoustic therapy (VAT) and, therefore, WBV studies cannot be mixed with VAT in the examination of mechanisms. First, although many applications of WBV use frequencies in the level of infrasound (1-15 Hz) they also use higher frequencies (e.g., 20-40 Hz) in the same range that VAT frequently uses. Music stimulation and VAT can employ pulsed sound units at 1-15 Hz and, in fact, "rhythm" in music is primarily in that frequency. So, there is no set frequency that makes it one or the other. Secondly, the axis of the applied vibration may be a discriminator between approaches (e.g., vertical (axial), horizontal, or multidirectional) but at the mechanism level there does not appear to be strict differentiation. For example, with blood flow slow axial (direction of the spine) WBV creates pulsatile stress on endothelial cells and so enhances blood flow at 1 or 2 Hz [ 33] but also with sound stimulation at 50 Hz [ 34]. Rhythmic driving of oscillatory coherence happens with rhythmic pulses at 1 or 2 Hz (delta entrainment, e.g., RAS effects) and at 10 Hz (alpha), 20 Hz beta, or 40 Hz gamma, etc. Consequently, at the level of the mechanism, WBV and VAT will be considered within the same domain of pulsed stimulation.

1.4. Methodology and Scope

1.4.1. Narrative Review Approach

In the next section we will present a narrative review of research literature that tries to map the basic "lay of the land" of vibration stimulated mechanisms and what health conditions may be affected. This is not a systematic critical review of research methodology, but rather a "cataloguing" effort to describe applications of vibration and mechanisms of effect. This effort was motivated by the perception that this has not been done to date. Researchers working on the mechanisms of vibration effect have essentially worked in silos. We determined to look for commonalities among the silos by looking not at the vibratory device, frequency, or context of application, but rather at mechanisms involved. Consequently, our search for studies to include was an emergent process from general to specific with the intent to identify categories of mechanism and then the specifics within the categories. We used a basic strategy to locate candidate studies: an electronic database search including PubMed, Scopus, and Google Scholar. Initial searches included terms such as "Vibration and Health", "Vibration mechanism", and "Vibration treatments" from years 1975 to 2021. We filtered based on number of citations and considered the more cited work first. After an initial collection of literature and, as researchers starting with the greatest awareness of VAT where the 1996 FDA claims for blood circulation, pain reduction (neurological), and mobility (musculoskeletal) effects were established, we decided to focus on three central concepts of vibration mechanisms for further searches: the effects of vibration on the blood, the nerves/brain, and bone/muscle. A further refined search included terms like "vibration and blood mechanism", "vibration and neuron mechanism", "vibration and brain", "vibration and bone/muscle mechanism", etc., followed by specific mechanism like endothelial stimulation or mechanoreceptor response. The identification and selection of papers was driven primarily by an attempt to describe the "field", the "lay of the land" of vibration research. Further, our focus was closely held to mechanism and not evidence-based clinical research related to vibration without attention to mechanism. Papers were chosen based on whether we believed them to add to our understanding of the effects of vibration on health via those areas.

1.4.2. Mechanisms of Response to Vibration

The auditory and vibrotactile stimulation from low frequency sound shows effects that are essentially the result of two categories of mechanisms: (1) physical, through muscular and cellular means, and (2) neurological, through sensory-based stimulation of nerves and receptors. At the physical level sound vibration is sensed by tactile receptors in the outer skin (Merkel disks-sensing vibratory strength and responding most to 5-15 Hz), inner skin (Meisner corpuscles-sensing vibratory frequency and responding most to 20-50 Hz), and in deeper tissues (Pacinian corpuscles-sensing acceleration and responding most to 60-400 Hz) [ 35, 36]. To avoid numbing of these sensors, VAT is usually constantly varied in amplitude (power pulsation) and/or frequency (scanning). A physical therapeutic effect can be obtained at a cellular and lymphatic level due to increased fluid and cellular waste transport, increased cellular metabolism [ 37, 38], increased blood circulation, and muscular relaxation due to a resonance response. Within the brain, vibration hypothetically enhances flow of cerebrospinal fluid and speeds removal of metabolic waste [ 39]. Most research with VAT has not explored neural oscillatory effects but recent studies show [ 40, 41, 42, 43, 44] potential brain effects, especially through prolonged application of a single frequency (e.g., 40 Hz).

1.5. Organization of the Analysis of Mechanisms

In the following three sections we explicate three primary categories of mechanisms activated by vibration applied to the body: hemodynamic, neurological, and musculoskeletal. By using the concept of mechanism established by Craver and Bechtel [ 5] we consider the causal and organizational dimensions to identify what we call basic mechanisms and submechanisms. This is a utilitarian simplification to focus, what is in most cases a complex system, on the organizational sequence. For example, vibration results in stimulation of endothelial cells (basic or first step mechanism), which then in turn releases nitric oxide and adrenomedullin, which have different effects and so are designated submechanisms. As we have stated earlier, this will not be an exhaustive and definitive description of complex chains of mechanistic actions. Rather we are setting out to point to directions for further research investigations of the effects of vibration on the body. In addition to identifying basic causal mechanisms, we describe some health related applications. Not every mechanism described in research has clear clinical applications. This is a nascent field where much more research is required and where knowledge translation into clinical application is needed.

2.1. Basic Mechanism: Stimulation of Endothelial Cells

2.1.1. Submechanism: Nitric Oxide

One of the effects of vibration applied to the body acknowledged by the FDA in 1996 was an increase in blood circulation [ 28]. The question is by what mechanism does vibration affect blood flow. A crucial signaling molecule that has a pivotal role in regulating blood flow and oxygenation of tissues is nitric oxide (NO). Nitric oxide is produced and released into the blood by the endothelial cells that line the blood vessels and lymphatic vessels. Vibration stimulates the endothelial cells to produce and release NO in several forms of which the isoform endothelial nitric oxide synthase (eNOS) is of particular importance for the generation of NO from endothelial cells. Although the detailed mechanisms of how sound affects the mechanosensors in the endothelium to control eNOS is not yet completely understood, it appears that the endothelial cells mechanosensor-proteins, Syndecan-4 (Syn4) vascular endothelial growth factor (VEGF), and Krüppel-like Factor 2 (KLF2) translate the physical force from the vibration into biochemical signals. Studies have shown that vibroacoustic stimulation can induce Syn4 and VEGF [ 50, 51]. NO regulates blood flow and vascular tone by affecting the vascular smooth muscle with the activation of the enzyme guanylate cyclase (sGC) [ 52] and the phosphorylation of extracellular signal-regulated kinase (ERK1/2) [ 53]. Although the purpose of stimulation, mode of vibration delivery, and frequency of vibration varies, endothelial cell stimulation releases NO and induces blood flow. The effect has been observed with whole body periodic acceleration using a platform (pGz) at 2 Hz [ 54], with microcirculation in the skin at 47 Hz [ 55], externally applied vibration to the arm at 50 Hz [ 34], sonic vibration applied to the chest at 100 Hz [ 56], and at various body surfaces at 150-250 Hz [ 57].

Application: General Blood Flow

Since vibrational stimulation induces blood flow, this may find general application to conditions resulting in decreased blood flow like diabetes. Maloney-Hinds [ 34] used 50 Hz vibrational stimuli for five minutes on participants' forearms and found NO production increased by 374% in the healthy controls and by 236% in the diabetes group. Skin blood flow was significantly increased (p = 0.0001) in both groups. Johnson et al. [ 58] studied diabetes patients with whole body vibration at 26 Hz and also found significant increase (p = 0.01) in skin blood flow but with less effect than Maloney-Hinds using direct to skin vibration.

Application: Cardio Protection and Therapy

An approach to stimulation of endothelial cells is "periodic acceleration" (pGz) consisting of repetitively moving the body in a head-to-foot direction at a typical rate between 1-3 Hz resulting in an increase in "pulsatile shear stress" on the endothelium-the friction of the blood against the endothelial lining of blood vessels resulting from the "pulse" of the movement and the gravitational force on the blood. This "shear stress" of the blood against the lining stimulates the release of NO and other endothelial-derived cytoprotective factors.

One application of pGz is as a form of cardiopulmonary resuscitation (CPR) where the body is moved head to foot at a rate similar to standard compression CPR. Adams et al. [ 59], in an animal study, found pGz as effective as standard CPR in terms of resuscitation survival rate and other acute outcomes but with fewer rib fractures.

An animal study by Adams et al. [ 60] applied pGz at 3 Hz for 30 min after induced cardiac arrest and resuscitation. The animals receiving the pGz treatment had a smaller reduction in the function of heart contraction (myocardial stunning), greater flow of blood to the heart, kidneys, stomach, and brain, showed less of an increase in Troponin I, a protein released into the blood stream indicating damage to the heart muscles, and a greater increase in eNOS.

The previous two studies by Adams et al. [ 61] used endothelial stimulation as a post-cardiac arrest treatment. Does it also offer preventative protection? They examined this by studying the occurrence of tissue damage caused by a return of oxygen rich blood following a period of oxygen deprivation (ischemia-reperfusion injury) caused by ventricular fibrillation (VF). Animal subjects were given one hour of pGz stimulation at 3 Hz prior to the induced VF. The animals receiving the stimulation had fewer arrhythmias of the blood circulation pulse, less myocardial stunning, stronger regional blood flow to organs of the body, and less indication of heart damage (Troponin I). Tests showed that the animals receiving the pulsed stimulation at 3 Hz had higher levels of endothelial nitric oxide with the conclusion that elevation of eNOS may have a cardioprotective function.

The studies by Adams reported above focus on heart function and effects where blood volume is constant. Bassuk et al. [ 62] looked at the effect of one hour of pulsed stimulation (pGz at 3 Hz) before and during significant blood loss from hemorrhage. The results showed significantly less oxygen consumption in the treatment group at all points of the hemorrhage and, after 40 mL/kg of blood loss, better preservation of regional blood flow to stomach, ileum, kidneys, heart, and brain. The authors attribute this effect to the shear stress produced by the motion on the endothelium and the release of nitric oxide.

Application: Neuroprotection and Neurotherapy

Aerobic exercise and the pulsed stimulation of whole body periodic acceleration (WBPA), also known as pGz, both have been shown to have positive cardiovascular effects through activation of nitric oxide (NO). In addition, the NO effect is known to induce the brain derived neurotrophic factor (BDNF) and glial derived neurotrophic factor (GDNF) thereby contributing a neuroprotective (prior to damaging events) and neurotherapeutic effect (following damaging events). A study by Adams et al. [ 33] showed that aerobic exercise significantly increases blood flow to the skeletal and heart muscles but not to the brain while WBPA increase blood flow with BDNF and GDNF to the brain and the heart but not to the muscles. They concluded that rhythmically pulsed stimulation of the endothelium can be a non-invasive strategy for neuroprotection and neurotherapy.

In a more detailed examination of the effects of WBPA on the brains of mdx mice, Lopez et al. [ 63] found an overload of calcium and sodium ions and an overproduction of reactive oxygen species (ROS) in the neurons resulting in cognitive dysfunction. After WBPA for one hour per day for eight days at 8 Hz, results showed that the elevation of calcium and sodium ions and the overproduction of ROS had been mollified along with an increase in enzymes that protect cells. This study again demonstrated the efficacy of pulsed stimulation of the endothelium to release nitric oxide and a cascade of factors that result in neuroprotection and neurotherapy.

2.1.2. Submechanism: Adrenomedullin

In addition to the release of nitric oxide, the vibratory stimulation of endothelial cells has been shown to release the cell protecting mediator, adrenomedullin (AM). It may function as a hormone in controlling circulation and vasodilation but also serves to stimulate angiogenesis-the growth of new blood vessels-and combats oxidative stress in cells. In this way AM can have a positive effect in cardiovascular disease including hypertension, myocardial infarction, and chronic obstructive pulmonary disease. However, in its function to extend blood supply in cells it may be a negative factor in relation to cancer. Martínez et al. [ 64] conducted an animal study to examine the effect of one hour of pGz stimulation on AM and found that immediately after the stimulation blood pressure was significantly reduced (from 115 ± 10 to 90 ± 8) and AM level was significantly increased and remained so for 3 h (from 776 ± 176 pg/mL baseline to 1584 ± 160 pg/mL, p < 0.01).

2.1.3. Submechanism: Antioxidants

Many disease conditions are linked to oxidative stress. These include cancer, Alzheimer's and Parkinson's disease, diabetes, and cardiovascular conditions like high blood pressure, atherosclerosis, and stroke. Release of nitric oxide into circulation is known to have some antioxidant properties. Uryash et al. [ 65], in a study with mice with high oxidative stress, specifically looked for the effect of pGz on eNOS and antioxidants. The subjects were given pGz for an hour a day and tissue was tested after one, two, and four weeks. The pulsed stimulation resulted in significant expression of antioxidants including glutathioneperoxidase-1 (GPX-1), catalase (CAT), superoxide, and superoxide dismutase 1 (SOD1). It also decreased reactive oxygen species (ROS).

2.2. Basic Mechanism: Vibropercussion

One of the features of vibration is that it results in one material striking against another-whether molecule to molecule or cell to cell or bone to bone, etc. The essentially mechanical action can produce health-related effects.

Application: Blood Clot Dissolution

To study the effect of mechanically pulsating a blood clot Hoffman and Gill [ 66] conducted an in-vitro study (outside the body) placing a one-hour-old blood clot into a solution of the medication Heparin typically used as a blood thinner to treat heart attacks or angina. The vessel holding the blood clot was then vibrated at 50 Hz for 20 min with a very low amplitude to simulate the amount of vibration that might be transferred into the body from a surface vibration. The result showed significantly better clot dissolution with vibration (25%) versus no vibration (5%). Hoffman and Gill [ 67] then basically replicated the study but applying the vibration externally through a 4 cm slab of meat as simulation of human body structure and varying the vibration to 50 Hz applied in short bursts of one per second. In addition to Heparin, they did a sample with Streptokinase, a medication specifically designed to break down clots. They again found the vibration very significantly increased clot dissolution, and with greater effect in the Streptokinase group attributed to enhanced mixing and penetration (or in a sense "drug delivery") of the lytic agent, which was originally disposed from the clot.

Effects of sound vibration stimulation on the neurological system are many and wide-ranging with multiple complex mechanisms involved. Most of these mechanisms are not yet fully understood but we will point to how the mechanisms at a cellular level seem to function and are related to sound vibration.

3.1. Basic Mechanism: Protein Kinases Activation

One of the questions of neuroscience is whether there is a way to regenerate or repair neural damage from neurodegeneration, stroke, transected nerve ends, etc. Electrical stimulation [ 71] is one intervention that has shown some success in creating axonal regeneration and neurite outgrowth. Low frequency sound vibration also appears to have potential at stimulating neurite outgrowth and neuronal differentiation.

Koike et al. [ 72], motivated by the intent to find why music therapy might be useful for Alzheimer's disease (AD) patients, conducted a study to determine if vibratory sounds might enhance neurite outgrowth. They focused on an in-vitro examination of PC12m3 cells known to be sensitive to nerve growth factor (NGF) that induces differentiation of nerve cells and neurite extension. They looked specifically at the p38 mitogen-activated protein kinase (MAPK) activity that has been shown by research with electrical stimulation [ 71] to be a pathway to enhancing PC12m3 cell growth, and which also appears enhanced in AD. They found that vibratory sound in the 10-100 Hz range had a positive effect on neurite growth with the strongest effect being at 40 Hz whereas vibratory sound at 150 Hz and 200 Hz had little effect. They found that 40 Hz stimulation enhanced p38 MAPK activity indicating that the neural outgrowth they observed was induced through the p38 MAPK pathway.

Cho et al. [ 73] and Kim et al. [ 74] examined the effect of 40 Hz sonic vibration on cell differentiation of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs). hUC-MSCs can differentiate into various types including neurons. Cho et al. applied vibration to the cells continuously for 5 days; Kim et al. for 3 or 5 days. Cho determined the differentiated cells were neuron-related oligodendrocytes because they expressed microtubule-associated protein 2 (MAP-2), which is a marker of synapse plasticity, glial fibrillary acidic protein (GFAP) thought to maintain astrocyte structure, and myelin basic protein (MBP) a component of myelin structure. Cho determined that the mechanism mediating the effect of vibration on the UC-MSCs was the extracellular-signal-regulated kinase (ERK) pathway. Kim et al. similarly examined protein expression related to neural differentiation stimulated by the 40 Hz vibration. They found that the protein calponin 3 (CNN3) promoted functional neural differentiation.

Choi et al. [ 75] examined the effect of multiple frequencies of low frequency vibration on adipose tissue-derived stem cells (AT-MSCs). Adipose tissue is commonly referred to as body fat and AT-MSCs are among the most studied in recent research. Choi applied vibration to the AT-MSCs for four days at 10, 20, 30, and 40 Hz with specific focus on the mechanism of ERK. They found that 30 Hz was most effective at affecting neural differentiation of these specific stem cells.

3.2. Basic Mechanism: Nerve Stimulation

Evidenced-based research repeatedly shows positive clinical effects from the application of pulsed stimulation of the body. This applies in varied neurological conditions including cerebral palsy [ 76], multiple sclerosis [ 77], and chronic musculoskeletal pain [ 78]. The question is whether this results in some way from a stimulation of the nervous system and if so, how does that occur. How pervasive is a system to respond to vibration? In the living body transmission of sensory information depends on sensory neurons and mechanosensation at axonal terminals in peripheral nerves. Different types of sensing neurons include mechanosensors detecting external signals, proprioceptors receiving internal body signals, and many types of nociceptors that detect noxious body-threatening stimuli. Usoskina et al. [ 79] examined the molecular mechanisms activated in the process of cells detecting vibration. By observing calcium ion transients in the somata of neurons, they saw that neurons reliably detected every individual stimulus (e.g., each molecular compression in a sound wave) and then converted these into specific firing patterns in the nerves. Given this basic mechanism, we next look at several categories in which this is applied.

3.2.1. Submechanism: Sensitization of the Proprioception System

The body's proprioception system gathers and processes information about changes in the position of joints and limbs and, therefore, is strongly involved in the control of posture and movement. Proprioceptors are mechanosensory neurons in the skin (Merkel disks and Meissner's and Pacinian corpuscles), muscles (spindles), tendons (Golgi tendon organs), and joints. This proprioception system involving receptors, nerves, spinal cord, and pathways of the central nervous system terminates within the thalamus and cerebral cortex [ 36].

Application: Complex Regional Pain Syndrome (CRPS)

Gay et al. [ 81] postulated that CRPS type I may be caused by a sensory input-output mismatch that leads to motor programming disorganization in cortical structures. They hypothesized that enhancing proprioceptive feedback with vibratory stimulation would minimize pain and increase range of motion. The study by Gay et al. [ 81] applied sinusoidal vibration at 86 Hz to the hand and wrist of patients with CRPS for 20 min a day, five days a week for 10 weeks in addition to conventional rehabilitation sessions. The control group received only the conventional treatment. The results showed that pain severity was lower by close to 50% and range of motion improved by about 30% in the treatment group. They attributed this result to a reestablishment of sensory input-output consonance.

Application: Cerebral Palsy

The research of Katusic et al. [ 80] proceeded on the premise that proprioception is crucial to motor control and hypothesized that sound-based vibration can resonate through the body and enhance sensation of body position, location, and orientation. Further they accepted the premise of Delecluse et al. [ 82] that vibration could alter corticospinal cell and spinal motor neuron connectivity and that this stimulation of the proprioceptive pathways could rearrange motor control strategies resulting in better postural stability. To test this Katusic et al. [ 80] did a three month study with 89 children with spastic cerebral palsy (CP) randomized to a physiotherapy only group and to physiotherapy plus vibration group. The vibration treatment, applied with a mat they could lie on, consisted of 40 Hz sine waves for 20 min, two times a week for 12 weeks. The vibration treatment group improved significantly in both spasticity and in gross motor function.

Ko et al. [ 83] observe that vibration has recently been shown to improve proprioception and thereby balance and motor skills. They postulate that this may be because vibration stimulates muscles and tendons. To test whether whole body vibration at 20-24 Hz would affect sense of joint position, gait, and balance in children with CP, they randomized 24 children to physical therapy (PT) or traditional PT plus vibration for 20 min (3 min on, 3 min off) two times a week for three weeks. They found significant improvement in joint position sense and improvement in gait variables in the vibration group.

3.2.2. Submechanism: Vagal Nerve Stimulation

Application: Depression

Sigurdardóttir et al. [ 88] conducted a study with 38 people with depressive disorder (18 treatment, 20 control) using relaxing music with a specifically created low frequency sound track that activated a vibrotactile transducer at the abdominal level at the back of the chair in which they were seated. The premised mechanism for their intended effect was the activation of Pacinian corpuscles sending an afferent impulse in the vagus nerve to the regions of the brain associated with depression. The vibratory stimulation treatment was applied for 20 min in eight sessions over 3-4 weeks. The authors did not report what specific frequencies they employed but maintain that Pacinian corpuscles stimulated at 240 Hz have a maximal afferent output but afferent output occurs at any frequency below that. Although not a rigorously controlled study and not measuring changes in the vagal tone, the pilot study did find a reduction in depression scores in the treatment group and attributes this to stimulation of the vagus nerve and the central nervous system through the abdomen. A study by Braun Janzen et al. [ 89] that applied a very similar treatment also found a reduction in depression and anhedonia although it did not premise vagal stimulation.

Application: Rett's Syndrome

Rett's syndrome (RS) is a gene mutation-based neurological disorder mainly affecting females that involves brain stem immaturity [ 90] resulting in dysfunction of the autonomic nervous system. The resultant symptoms are numerous with many associations to the vagus nerve. These include breathing and cardiac function, speech and hearing-related communication, and movement control and seizures [ 91, 92]. Vagus nerve stimulation has shown positive results in reducing epileptic seizures [ 91] and along with auditory stimulation has been effective in increasing cortical strength and auditory function [ 92].

Bergström-Isacsson et al. [ 93] examined neurophysiological brainstem data and emotional response in 29 Rett's participants and 11 controls to six stimuli with music and low frequency vibration at 40 Hz. Each stimulus was presented for 10 min in a repeated measures design. Outcome measures used were mean arterial blood pressure, the coefficient of variation of mean arterial blood pressure, cardiac sensitivity to baroreflex, and cardiac vagal tone (CVT). Results showed that the strongest parasympathetic response in the Rett's group was to the 40 Hz vibratory stimulation and with a significant increase in CVT.

3.3. Category of Neurological Effects-Pain and Vibratory Analgesia Specific

The pain-reducing effect of vibration is demonstrated in numerous evidence-based research studies [ 78, 94, 95, 96, 97]. Mechanical and acoustic vibrations have been used extensively to address pain and is a treatment technique in orthopedics and low back pain [ 98, 99], physiotherapy [ 100, 101, 102], during cosmetic procedures [ 103], and during orthodontic work and orofacial pain [ 104, 105, 106, 107]. However, until relatively recently, very little was thoroughly described and understood about pain perception mechanisms. Studies with electrophysiology have shown cortical neurons responding to noxious stimuli, but it has not become clear to what extent this response represents pain or correlates with it [ 108]. Consequently, the mechanisms by which vibration acts as an analgesic is less understood.

3.3.1. Basic Mechanism: Gate Control

One of the submechanisms of the general mechanism of nerve stimulation but a basic mechanism of analgesia is focused on the function of the substantia gelatinosa in the dorsal horn and is commonly known as gate control. The theory for this pain mechanism was proposed by Melzack and Wall [ 109] and postulates that the substantia gelatinosa modulates sensory information being transmitted to the spinal cord and the brain. Specifically, signals from pain receptors are carried to the dorsal horn by small diameter afferent A-delta and C fibers. The signal transmission from the pain receptors can be modulated (inhibited) by the large afferent A-alpha and A-beta fibers transmitting sensory signals from skin sensation such as touch or vibration [ 78, 96, 109]. This mechanism known as gate control theory has been subject of considerable criticism and aspects have been questioned [ 110, 111] and enhanced by Melzack's neuromatrix of pain theory [ 112] but its fundamental function remains.

Salter and Henry [ 113] specifically explored the response of wide dynamic range (WDR) spinal neurons in the lumbar dorsal horn to vibration at different amplitudes and frequencies to determine how they might play a role in analgesic effects. They tried a variety of frequencies and intensities and found that WDR neurons entrained to the vibratory frequencies below 80 Hz. Their findings suggest that pain reduction is accomplished by the effect of vibration on Pacinian corpuscle afferents and the WDR neuron response in the dorsal horn but that frequency, location, and intensity of the vibratory stimulus is a factor that needs further clarification.

3.3.2. Basic Mechanism: Modulating Autonomic Responses-Pain and Beyond

An alternative medical practice that has growing scientific evidence for its effect on the central nervous system and regulating autonomic responses is acupuncture [ 114]. The clinical efficacy of acupuncture may reside in its neurobiological foundations and in its purported effect of creating biochemical changes in the body and brain that control the autonomic nervous system's functions such as blood pressure, heart rate variability, skin temperature, and perception of pain. The detailed explanation of mechanisms is still needed.

3.3.3. Basic Mechanism: Neurotransmitters-Pain and Beyond

Unpacking the complexity of the role of neurotransmitters and neuromodulators on the body and brain is beyond the scope of this paper. However, research has shown instances in which neurotransmitters are stimulated by vibration and these will be described here.

In relation to vibratory analgesia, Salter and Henry [ 118] examined specifically the vibratory activation of P ₁-purinergic receptors in the dorsal horn by the neurotransmitter adenosine. In a study with cats, vibration was applied at 80 Hz in 2.5-3.5 s trains every 20-25 s for 10 min. This resulted in a vibration-induced depression of lower lumbar nociceptive neurons and remained in effect for up to 4 h after the stimulation. Various agents used to attenuate the depression of these neurons revealed that adenosine was responsible for the analgesic effect. The study suggests that the effect earlier described as gate control may be mediated by the release of adenosine resulting from vibration.

Several neurotransmitters not specifically related to pain have already been discussed in other contexts above. Assuming vibration can stimulate the vagus nerve and the related splenic nerve as discussed above, vibration then has the effect of releasing acetylcholine, which plays a key role in synapses and especially at the location where nerves and muscles connect. It also plays a role in controlling the autonomic nervous system and a particularly important role in the cognitive system and in regulating heart rate. Additionally, stimulation of the splenic nerve causes the induction of norepinephrine. Nitric oxide, extensively discussed above, is also a neurotransmitter serving to regulate and mediate processes of the nervous, immune, and cognitive systems.

Gamma-aminobutyric acid (GABA) is a prominent neurotransmitter in the brain and the central nervous system. It plays a crucial role reducing the activity of neurons and especially in control of fear and anxiety. Safarov and Kerimov [ 119, 120] in two separate animal studies explored the effect of low frequency vibration (20 Hz) on GABA levels and its metabolism. The found that vibration, regardless of duration, increased GABA level in the brain stem, the large hemispheres, and the cerebellum as well as activity of the glutamate decarboxylase enzyme, which produces GABA. However, they found the effect more marked with 30 min stimulation than with long periods like 7 h. The implication is that one of the mechanisms creating the relaxation effect of vibroacoustic stimulation may be the boost in neurotransmitter GABA.

3.4. Basic Mechanism: Oscillatory Coherence Supports Connectivity and Circuit Function

The neurological effect from rhythmically pulsed sensory stimulation is premised on two important postulates: (1) that rhythmic sensory stimulation (RSS) drives a neural response resulting in increased oscillatory coherence and (2) that oscillatory coherence is crucially linked to connectivity, circuit function, and related to health conditions.

As to the postulation that RSS drives a neural response, recent research in somatosensory, auditory, and visual modalities shows that vibrotactile rhythmically pulsed stimulation has a strong neural driving effect [ 121, 122, 123]. For example, vibratory stimulation of a finger, the hand, or the median nerve results in an oscillatory response in primary and secondary sensorimotor cortices [ 124, 125, 126] and attention plays almost no role [ 127]. More auditory rhythmic stimulation research has been done to elicit steady-state or spontaneous oscillatory responses using clicks, amplitude-modulated isochronous sounds [ 128], or pure tones. Examples include a 40 Hz amplitude modulated tone [ 129, 130], or even the rhythms of binaural beats that are created through binaurally detuned tones [ 131]. What follows then is the conclusion that RSS can drive oscillatory coherence.

The assertion that oscillatory coherence is linked to circuit function and related to health conditions is more crucial but less understood. Deep brain stimulation (DBS) research suggests that circuit dysfunction is common to many psychiatric and neurological conditions [ 132, 133]. Basically, the dysregulation of circuits that underlies these conditions stems from a lack of excitation-based coherence, disturbed coherence, or coherence that is overly strong in inappropriate neural populations. Llinas pointed specifically to the recurrent connections between the thalamus and the cortex that have a mechanism function to connect areas of the cortex and control information flow [ 133, 134, 135, 136, 137]. These thalamocortical loops serve communication in the brain similar to what an internet hub does. Llinas further maintained that thalamocortical interconnectivity depends primarily on rhythmic oscillatory activity. Thalamocortical loops function optimally with rhythmic oscillatory activity in the cortex in the gamma band (40 Hz) and in the thalamus in the alpha band (10 Hz). Thalamocortical dysrhythmia (TCD) is characterized by a decrease in alpha band activity (power) with a related increase in theta band activity (4-7 Hz) and a reduction in cortically consistent gamma band activity. TCD appears related to neurological and psychiatric conditions; specifically cognitive, motor, auditory, and mood functions. TCD is linked to conditions including Parkinson's disease, major depression, neurogenic central pain, tinnitus, and schizophrenia [ 134].

Assuming then that vibratory stimulation (RSS) can drive oscillatory coherence and potentially regulate dysrhythmic circuits in the brain, RSS may employ the mechanism of oscillatory coherence and positively affect the health conditions resulting to some extent from these dysrhythmias. The positive response of major depression to RSS may be an indicator of this [ 89]. The mechanism of driving oscillatory coherence with RSS broadens the focus from the neuroscience of circuit connections (the connectome) to the framework of dynamic brain rhythms related to neural spiking activity (the dynome) [ 13].

3.4.1. Application: Neurogenic Pain

In this context where FM seems to be related to brain dysregulation and connectivity, where pain appears to be related to brain region interaction, and where there is demonstrated potential for vibration to drive neural coherence, it can be speculated that the positive results from vibratory stimulation on FM patients were due to the mechanism of oscillatory coherence [ 30, 140]. Both studies [ 30, 140] used 40 Hz vibrotactile stimulation of the body, used oscillatory coherence and its relation to connectivity as a premise for effect, but did not establish mechanism with brain imaging. The authors are currently engaged in a study to explore this mechanism.

3.4.2. Application: Neurodegenerative Conditions

The specific mechanisms or causes related to neurodegenerative diseases and conditions are not fully understood but one path being explored with some success is that of spontaneous brain oscillation power, synchronization, dysrhythmia, and circuit connectivity.

Dockstader et al. [ 141] studied the oscillatory power in various frequency bands in brain regions using magnetoencephalography (MEG) imaging in children who had been treated for brain tumors with cranial radiation therapy (CRT). Children treated with CRT consistently are found to have cognitive deficits in attention, processing speed, and memory. The results showed a clear pattern of gamma deficits with a significantly lower gamma oscillation power in the 60-100 Hz range in the resting state.

Stam et al. [ 142] studied Alzheimer's patients (AD) and healthy controls with full-head MEG with the purpose of investigating linear and non-linear interdependencies in the MEG signal channels. Results showed that this measure of synchronization was lower in AD in the 10-14 Hz alpha band, 18-22 Hz beta band, and in the 22-40 Hz gamma band. Additionally, studying AD patients with MEG, Ribary et al. [ 136] looked at 40 Hz oscillatory coherence at cortical and subcortical sites associated with the thalamocortical loop. Results showed that although AD patients had similar activity patterns as healthy patients, the AD patients had reduced gamma activity in the cortical component.

Lozano and Lipsman [ 132] review and explain dysfunctional brain circuits and specifically how deep brain stimulation (DBS) can mitigate the effect, for example, of the motor circuit affecting Parkinson's disease (PD). The role of DBS in relation to the dysfunctional motor circuit in PD is the inhibition of a group of neurons with 130 Hz electrical stimulation. While the widely accepted approach of DBS is an inhibitory one, Neuman et al. [ 146] point out that DBS modulates the motor circuit but does not restore it functionally. They differentiate the motor circuit pathways more precisely in the basal ganglia and suggest their work may inspire innovative ways to improve the therapeutic efficacy of neuromodulation in PD. Several studies have used sound vibration as a neuromodulatory stimulant with PD patients [ 44, 147]. One study applied 30 Hz and the other 40 Hz. Both had significant positive results and, although no neuroimaging was used to verify effects on circuit function, they pointed to the potential mechanism of vibration therapy with PD.

3.4.3. Submechanism: Frontal Oscillatory Symmetry

The discussion above has already identified a vibration mechanism related to major depressive disorder (MDD) under vagal stimulation and under oscillatory coherence and TCD. Decreased connectivity is a clearly identified factor in MDD [ 148] and so affecting MDD by driving coherence with rhythmic sound and vibration is a possibility. There is a further potential mechanism for MDD related to frontal asymmetry. There is considerable evidence that frontal EEG asymmetry between left and right in the alpha band is a biomarker for depression and anxiety [ 149] but age, gender, and severity of depression interaction raises a caution on this [ 150]. However, studies have shown that using music stimulation with depressed participants has resulted in a reduction of frontal asymmetry in the EEG [ 151, 152, 153]. These music studies did not use low frequency sound or vibrotactile stimulation. The only known study that has used vibrotactile gamma stimulation [ 89] did show significant reduction in depression severity. More research into the occurrence of frontal oscillatory asymmetry and vibratory stimulation is needed.

Effects of sound vibration stimulation on the musculoskeletal system are many and wide-ranging with multiple complex mechanisms involved. These structures include the muscles, skeleton, intervertebral discs, ligaments, and other associated structures. We will point to how the mechanisms at a cellular level seem to function and are related to sound vibration and how it translates to applications in humans.

4.1. Basic Mechanism: The Muscle Stretch Reflex

The physiological basis for vibration acting on the muscles involves the mechanical stimulation of the muscle stretch reflex leading to neuromuscular potentiation [ 154]. The reflex functions to maintain a constant muscle length so any stretches result in involuntary muscle contractions, and so a low frequency (0-200 Hz) paradigm of vibration can lead to thousands of such muscle contractions within minutes of application [ 155]. Vibration of the muscles causes a cascade of events: afferent neurons stimulate alpha motor neurons, leading to motor unit recruitment, increased firing frequency, and/or improved synchronization, which leads to quicker or more forceful muscle contraction and an overall increase in muscle fibers over time (i.e., hypertrophy) [ 154]. Muscle hypertrophy is correlated with an increase in protein synthesis and an addition of contractile filaments, leading to greater muscle strength. The Akt/mTOR/p70S6K signaling pathway in muscle cells is a crucial component in the hypertrophy process and is necessary in inhibiting the opposite (i.e., muscle atrophy), and has been shown to be enhanced by vibration stimulation in both mechanical loading and muscle injury contexts [ 156]. Several studies have shown that Akt levels increase in response to muscle contractile activity and mechanical tension, both of which are stimulated by vibration treatment [ 155, 157].

Vibratory stimulation of muscle cells in vitro and in vivo also decreases the expression of genes that prevent atrophy: namely myostatin and atrogin-1 [ 158, 159]. Myostatin regulates muscle growth by inhibiting myogenesis and atrogin-1 enhances proteolysis functions. The decrease in myostatin and atrogin-1 after 30 Hz vibration treatment has been associated with the fusion of satellite (muscle) cells [ 155].

The muscle hypertrophy pathway and atrophy pathway are not completely independent however, and function antagonistically to each other. For example, when Akt is activated it phosphorylates FOXO1 and sequesters it in the cytoplasm, inhibiting its transcriptional activity [ 156]. Without Akt activity, FOXO1 can induce transcription of atrogin-1, which leads to atrophy via increased protein degradation [ 160, 161]. Vibration may also enhance mitochondrial biogenesis, which normally occurs as a major adaptation of skeletal muscles in response to exercise training. One study found that vibration at 50 Hz enhanced the expression of PGC-1α in the soleus muscle, gastrocnemius muscle and liver, and was associated with increased muscle strength [ 162]. PGC1α plays an important role in mitochondrial biogenesis and is regulated by mitogen-activated protein kinase p38 (p38 MAPK) and is activated during exercise.

Application: Using Vibration to Provide Exercise Like Effects

Gloeckl et al. [ 161] and Lage et al. [ 162] were able to safely and successfully use vibration therapy as a pulmonary rehabilitation tool for patients with chronic obstructive pulmonary disease (COPD) who require physical exercise but cannot perform due to breathing limitations and general malaise. Although physical exercise is the basis of pulmonary rehabilitation for people with COPD, intense exercise may also stimulates pro-inflammatory cytokines and can be harmful in that context [ 165, 166]. Since the magnitude of the exercise stimulus is related to the increase in inflammatory cytokines, treatment for COPD needs to stimulate the muscles without such complications [ 167]. For this reason, clinical studies have tested vibration therapy for COPD and found functional improvements in mobility and an anti-inflammatory effect [ 168, 169].

Another application has been in the use of vibration treatment for frail elderly people. Sarcopenia is a form of muscle loss that occurs with aging and/or immobility. Strength training can reverses age-related muscle and strength losses while promoting muscle hypertrophy [ 170]. However, due to the intensity of exercise needed to achieve such a result the appeal and application by the elderly is very restricted, with only 10-15% of this population reported to engage in such training [ 171]. Vibration treatment of the elderly is thus a safe and effective strength training tool that has shown high compliance among the elderly population and has shown positive results in improving in balance, physical function, and muscle strength [ 172, 173, 174].

A third condition for which vibration may be applied is for Duchenne muscular dystrophy (DMD), which is a degenerative disorder caused by a defective gene responsible for producing a muscular protein called dystrophin. This protein plays an important role in preventing muscle fatigue, and patients with DMD often have poorly developed muscles and, as a consequence, bone. Exercise is a crucial treatment for muscle and bone growth; however, over-activity among DMD patients can result in pain, myoglobinuria, and further fatigue in the muscles [ 175]. Therefore, vibration treatment can act as a useful adjunct to exercise therapy to keep muscle fatigue low while maintaining optimal growth. Moreira-Marconi et al. (2017) found that vibration treatment can improve or maintain functional mobility and strength in the muscles among DMD patients [ 176]. In summary, the benefits of vibration on the muscles present an important modality for treatment that includes a low risk, high compliance form of stimulation that may benefit a wide variety of conditions affecting the musculature.

4.2. Basic Mechanism: Determining Bone Cell Progenitor Fate

The skeleton is a very dynamic structure that is maintained by bone deposition and resorption, determined largely by the activity of osteoblasts and osteoclasts respectively. Structurally, bone is created of different types of cells and a rich extracellular matrix (ECM). Osteoblasts have the ability to secrete and calcify the ECM and are derived from mesenchymal stem cells (MSCs). The osteogenic differentiation of MSC's involve three stages: commitment to osteogenic lineage, matrix synthesis, and matrix mineralization [ 177]. The major genetic markers for osteoblasts include RUNX2, ALP, Col-I, OSX, and OCN.

(1)

RUNX2: a protein encoded by the RUNX2 gene that is a key transcription factor associated with osteoblast differentiation.

(2)

ALP: alkaline phosphatase. An enzyme responsible for breaking down proteins and is associated with bone growth.

(3)

Col−1: collagen type 1. It is main structural protein in the extracellular matrix found in the body's various connective tissues.

(4)

OSX: Osterix, also called transcription factor Sp7. It plays a major role in driving the differentiation of mesenchymal precursor cells into osteoblasts and eventually osteocytes.

Zhou et al. (2011) investigated the effect of vibration on the osteogenic differentiation of MSCs seeded on human bone-derived scaffolds and found that 40 Hz vibration promoted MSC differentiation by upregulating the mRNA and protein expression of RUNX2, ALP, Col-I, and OCN [ 178]. Zhang et al. (2012) cultured periodontal ligament stem cells under vibration and in the 40-120 Hz range found increased levels of ALP, Col−1, Runx2, Osx, and OCN [ 179]. Additionally, Prè et al. treated MSCs with mechanical vibration, and the results showed that the expression of ALP and Runx2 was significantly increased after 30 Hz mechanical vibration treatment (2017) [ 180]. This suggests a direct role in vibration in influencing the stem cell fate toward osteoblast formation.

RUNX2 and OSX are usually highly expressed at the early stages where stem cells commit to the osteogenic lineage [ 181, 182], Col−1 and ALP at the middle stage (matrix synthesis) [ 179, 182], and OCN at the late stage of osteogenesis [ 183]. RUNX2 and OSX are transcription factors that are important for stem cell commitment to the osteogenic lineage and are expressed specifically in osteoblasts. RUNX2 are active earlier and OSX at a later step once preosteoblasts form into fully functional osteoblasts. Studies looking at molecular and genetic manipulation of RUNX2 in vivo found that its expression is necessary for MSC differentiation towards the osteoblast lineage [ 184, 185], and without it this process cannot begin [ 186]. RUNX2 is also known to negatively regulate osteoblast proliferation by acting on the cell-cycle [ 181], and prevents terminal differentiation of osteoblasts to keep them in an immature state. This corresponded with several studies that found that the proliferation of osteoblasts did not change or was decreased after vibration [ 187, 188, 189]. OSX is a more specific marker of osteoblasts and is expressed after RUNX2, and further guides differentiation from preosteoblast to immature osteoblast state [ 182]. OSX binds to the nuclear factor of activated T cells (NFAT), which leads to the expression of collagen type 1 and activation of the Wnt signaling pathway [ 190]. Chen et al. (2016) found that vibration treatment of MSCs cultured on hydroxyapatite-coated surfaces increased in the expression of Wnt and β-catenin in addition to RUNX2 and OSX [ 177]. The Wnt/β-catenin signaling pathway is a normal physiological response to mechanical loading, which enhances the sensitivity of osteoblasts to further mechanical loading [ 191]. Hou et al. (2011) investigated the mechanisms of vibration-enhanced osteogenic responses in MC3T3-E1 cells and they demonstrated that Wnt signaling was involved in the transduction of vibrations, which caused a decrease in the RANKL/OPG ratio and levels of sclerostin [ 192]. The RANKL/RANK/OPG pathway is an important signaling pathway in the formation and activity of osteoclasts, and is one that is also influenced by vibration treatment. Osteoclasts are derived from monocytes and macrophages, which proliferate in the bone marrow and fuse to give rise to multinucleated osteoclasts [ 193]. Osteoclast progenitors express RANK (receptor activator of nuclear factor-κB), which interact with the RANK-ligand (RANKL) to initiate osteoclastogenesis. RANKL is produced by a variety of cells such as osteoblasts, osteocytes, stromal cells, and lymphocytes. Osteoprotegerin (OPG), on the other hand, binds competitively to RANK and inhibits this process. Therefore, the availability and interaction of RANKL/RANK/OPG determine the rate of osteoclastogenesis [ 194]. Therefore, the Wnt/β-catenin signaling pathway stimulated by vibration produces a dual function to stimulate the formation of osteoblasts while inhibiting the formation of osteoclasts. In other words, vibration stimulated an increase in bone amount and quality.

Kulkarni et al. (2013) found that mechanical vibration of osteoclast precursor cells reduce DC-STAMP expression in the presence of RANKL in osteoclast precursor cells leading to the inhibition of osteoclast formation [ 195]. Wu et al. (2012) found that stimulation of murine monocytes by vibration reduced the expression of osteoclast-specific genes such as cathepsin K, matrix metallopeptidase-9 (MMP-9), and c-Fos to such a level that cells could not differentiate into osteoclasts even in the presence of RANKL [ 196]. Kim et al. (2012) and Sun et al. (2011) independently found that vibration stimulation of mesenchymal stromal stem cells increased OPG expression, indicating a direct effect of vibration in inhibiting osteoclast formation [ 197, 198].

In summary, it seems that vibration stimulation on bone related stem cells promote anabolic processes by stimulating the formation of osteoblasts while simultaneously inhibiting catabolic processes by inhibiting the formation of osteoclasts.

Application: Tissue Engineering

A major application of vibration in the differentiation of stem cells in the context of bone health is in engineering bone tissue. Typically, this includes osteoblasts and other bone cell progenitors growing and attaching to an artificial surface for eventual implantation. The ability of MSCs to adhere to the implant surface and their differentiation on the implant surface are important components of successful osseointegration [ 199, 200]. In order to analyze cell adhesion to the implant surface, Chen et al. (2016) assessed the matrix organization and the cytoskeleton rearrangement (F-actin), and the gene expression of F-actin, β1 integrin, vinculin, and paxillin, which are involved in the adhesion of cells to substrates. They found that vibration of MSCs on hydroxyapatite-coated surfaces significantly increased the expression of F-actin, and such upregulation of F-actin by mechanical stress has also been reported in other studies [ 201, 202]. Mechanical strain is known to induce changes in cytoskeletal organization, and actin filaments are crucial for cell adhesion [ 203]. Chen et al. (2016) also found an increase in other proteins necessary for adhesion such as β1 integrin, vinculin, and paxillin [ 177]. This has been found in other studies as well [ 201, 204]. In summary, the differentiation of MSCs to anabolic processes and the adhesion to surfaces after vibration allow for this to be an ideal application for engineering bone tissue in the lab for implantation.

4.3. Basic Mechanism: Vibration Effects on Ossification and Resorption

Bone remodeling is a lifelong process where mature bone tissue is removed (i.e., bone resorption) and replaced by new bone tissue (i.e., ossification). This process is important for the adaptation of bone to mechanical loading and healing from microdamage and fractures. The imbalance of bone resorption and ossification leads to disorders such as osteoporosis, a systemic skeletal disorder characterized by the deterioration of bone tissue leading to bone fragility and an increase in fracture risk. Bone mineral density (BMD) is a measurement of the amount of bone mineral in bone tissue, and is used clinically to assess the risk to osteoporosis or fracture. In this section we lay out the evidence that vibration treatment has anabolic effects on bone remodeling, and how this translates to better bone health and BMD when applied to humans.

Healthy bone remodeling involves maintaining a balance between bone resorption and ossification and is maintained by a network of regulatory proteins [ 205]. Bone resorption is done via the action of osteoclasts that break down bone, and bone formation (i.e., ossification) is mediated by osteoblasts, which secrete new bone material. As described in Section 4.2, vibration stimulation has been showed to shift the balance towards bone formation by enhancing anabolic pathways and inhibiting catabolic pathways. Osteocytes (i.e., mature bone cells) are the major mechanosensor in bone that influence osteoblast and osteoclast activities when subjected to a variety of mechanical stimuli, including fluid flow, hydrostatic pressure, and mechanical stretching [ 206].

Animal models have shown that vibration at 10-100 Hz can stimulate bone growth by doubling bone formation rates and inhibiting osteoporosis [ 207]. Bacabac et al. (2006) specifically tested the effects of vibration on osteoblast precursor cells at a variety of frequencies from 5 to 100 Hz. They found that nitric oxide (NO) release and COX-2 mRNA expression were positively correlated with frequency whereas prostaglandin E ₂ release negatively correlated with frequency [ 208]. It has been shown in vitro that osteoclasts migrate away from NO [ 208, 209], and PGE2 I stimulates osteoclasts differentiation and activity by increasing mRNA levels of osteoclast differentiation factors. Therefore, the anti-correlation of NO and PGE ₂ has a net effect of stimulating ossification over bone resorption.

Da Jing et al. (2016) tested vibration at 45 Hz and 0.5 g in leptin deficient mice, which exhibit decreased bone mass and impaired bone microstructure. They found that vibration treatment in these rats stimulate tibial alkalinephosphatase (ALP), OCN, runt-related transcription factor 2 (RUNX2), type I collagen (COL1), BMP2, Wnt3a, Lrp6, and b-catenin mRNA expression, and prevented the increases of tibial sclerostin (SOST), RANK, RANKL, and RANL/osteoprotegerin (OPG) gene levels indb/dbmice [ 210]. These proteins are pro-ossification and antibone resorption, and this ultimately resulted in increased mineral apposition rate, bone formation rate, and increased osteoblast numbers in cancellous bone. These beneficial effects on bone cells are supported by Li et al. (2015), who tested ovariectomized rats with vibration (35 Hz at 0.25 g) [ 211]. They found that vibration upregulated the expression of BMP-2 and Runx2, activated the ERK1/2 signaling pathway, and consequently led to increased expression of OCN. These proteins are also pro-ossification. They also noted that the anabolic effect of mechanical stimulation was enhanced with the incorporation of resting period between loadings. These findings were replicated by Sun et al. (2019) who showed that MLO-Y4 osteocyte like cells stimulated by 45 Hz at 0.5 g promoted the secretion of prostaglandin E ₂ and OPG, and inhibited the secretion of tumor necrosis factor-α and RANKL [ 212]. Tanaka et al. (2003) found similar results testing osteoblasts in vitro with vibration [ 213]. Lau et al. [ 206] found that stimulation of MLO-Y4 osteocyte cells stimulated with low amplitude high frequency vibration (0.3 g; 30, 60, and 90 Hz) had a variety of antibone resorption effects, which varied with frequency. They found that osteocytes increased the expression of COX-2 at 90 Hz, while RANKL decreased most significantly at 60 Hz. Conditioned medium collected from the vibrated osteocytes cells inhibited the formation of large osteoclasts and the amount of osteoclastic resorption by 20% [ 206]. Similar findings have been found in other studies and have also found an increase on chondrogenic factors such as collagen type I and II, and have shown corresponding improvements of fractures from radiographs and histomorphometry [ 214, 215]. Computed tomography imaging evidence on fracture healing showed that ovariectomized mice undergoing 45 Hz vibration treatment demonstrated improved callus properties, with increased flexural rigidity and bone formation in addition to the cellular effects as mentioned above [ 216].

The mechanism behind the frequency dependency of osteocyte response to vibration is not well understood. At lower frequencies (<10 Hz), fluid flow may be what mediates vibration mediated effects, causing calcium oscillations in cells when vibration is applied [ 217]. Due to the viscoelasticity of cells, cells are less stiff and thus more deformable at lower frequencies. Such deformation caused by fluid shear stress requires contact stress acting primarily on the cell membrane [ 208]. Consequently, cilia, which are small hair like structures protruding from the cell membrane, are considered important structures for mechanosensation. Li et al. (2020) found that chemicals which damage primary cilia inhibited vibration-induced osteogenic responses (differentiation, mineralization, and maturation) in osteocytes, indicating that vibration-induced osteogenic effects are mediated through cilia [ 218]. At higher frequencies there is a chance that fluid stress is attenuated, and it is suggested that at frequencies above 10 Hz, the movement of solid intracellular bodies such as the nucleus may drive the cellular effects of vibration. Bacabac et al. (2006) found that the rate of acceleration generated by the vibration frequency was correlated with the amount of force acting on the nucleus of vibrated bone cells.

The effects of vibration may have differing effects on different bones of the body and depending on how vibration is applied. Pravitharangul et al. (2018) found that vibration of bone cells from the iliac had reduced resorptive protein expression but that effect was not found in mandible bone cells [ 219]. Interestingly, Rubin et al. (2004) showed that whole body vibration signal was maximally transmitted to the hip and spine of people in a standing position [ 220]. Komrakova et al. (2013) investigated various vibration parameters on fracture healing in mice and found that vertical vibrations of 35 and 50 Hz produced more favorable results than the higher frequencies and horizontal vibrations showed no positive or negative effects. Specifically, vertical vibrations (>35 Hz) improved cortical and callus densities, enlarged callus area and width, accelerated osteotomy bridging (35 and 50 Hz), and increased relative muscle weight (50 Hz) [ 221].

Estrogen levels may also influence whether vibration generates positive or null effects on bone cells. Haffner-Luntzer et al. [ 222] found that vibration applied at 45 Hz stimulation of osteoblast cells had either positive bone forming effects of gene expression or a null effect depending on the presence of estrogen. They found that bone cells increased COX-2 gene expression, cell metabolic activity, and cell proliferation after vibration in the absence of estrogen, whereas the contrary was true in the presence of estrogen [ 222].

This indicates that considerably more research is needed to determine the optimal frequency, amplitude, and direction of vibration for desired bone effects. As well, specific important contextual considerations need to be explored to derive the intended effects of vibrations.

Application: Treating Osteoporosis and Bone Loss

An important application of vibration in humans is to stimulate bone healing and growth, especially in response to fractures or osteoporosis. Rubin et al. (2004) performed a prospective, randomized, double-blinded, and placebo-controlled trial of 70 postmenopausal women with brief periods of vibration applied at 30 Hz and 0.2 g [ 220]. DXA scanning was used to measure bone mineral density in the spine, hip, and distal radius at baseline, 3, 6, and 12 months. They found that vibration treatment significantly reduced bone loss in the spine and femur, with efficacy increasing with greater compliance and for those with lower body mass. Although bone loss was reduced, there did not seem to be any indications of a net increase in bone mass being created. In a more recent study, ElDeeb and Abdel-Aziem (2020) showed that vibration in conjunction with exercise can improve the bone mineral density (BMD) of post-menopausal women with osteoporosis [ 223]. Other clinical studies have shown similar results to suggest positive effects on osteoporotic BMD [ 224, 225].

Matute-Llorente et al. (2015) performed a randomized controlled trial of adolescents with Down syndrome using whole body vibration, with primary outcomes being bone mineral content and BMD [ 226]. Adolescents with Down syndrome (DS) tend to have poorer bone health than peers without DS. They found that the vibration treated group increased in bone mineral content and density as a whole and specifically in the lumbar spine area and the tibia. Another randomized, placebo controlled trial found that vibration treatment for children with cerebral palsy improved BMD in tibial regions and the spine compared to controls, even though compliance to using the device was only 44% [ 227].

Therefore, it can be seen that the beneficial osteogenic effects of vibration on bone health are prominent across different age groups and medical conditions with differing etiologies. What is common is the fact that osteoporosis, down syndrome, and cerebral palsy are disabling conditions that reduce mechanical stimulation of the bones due to a lack of activity or strength in muscles and bone. Since the nature of the vibratory stimulus is based on providing a surrogate for high frequency muscle based signals, this may be used successfully to treat conditions that limit such stimulation naturally. Given that the vibration parameters are constrained to 1-100 Hz at less than 1 g magnitude [ 228], there is a net beneficial effect of vibration on the bone strength and health in humans.

4.4. Basic Mechanism: Anabolic Effects on the Spine and Intervertebral Discs

The spine is an important component of the skeletal system that is crucial for structural integrity of the body and housing the spinal cord, which connects the peripheral nerves to the central nervous system. The spine consists of 33 vertebral segments, the upper 24 are articulating and separated by each other by intervertebral discs, and the lower nine are fused in adults. Each disc acts as a cushion-like joint between vertebral segments, allowing for slight movement while holding the segments together and acting as a shock-absorber. In this section the effects of vibration on the spine and associated structures will be explored.

The vertebral segments of the spine display the same effect from vibration as any other bone, in that there is an increase in BMD after vibration stimulation [ 23]. What is of great interest however is the effects of vibration on the intervertebral discs (IVD), as the degeneration of the intervertebral discs is considered as an important pathogeny for spinal disorders and low back pain [ 229]. Desmoulin et al. (2013) tested bovine IVD in multiples studies with 0-200 Hz vibration and found an increase in mRNA expression of aggrecan, collagen type I and II, decorin, and versican in significant amounts [ 230, 231, 232]. Another study found similar results in living mice treated with 15-90 Hz vibration at 0.3 g, showing an increase in the expression of aggrecan, biglycan, decorin, type I collagen, and Sox9 [ 233]. Liang et al. (2017) found that vertical and whole body vibration in rats (0.49 g at 30 Hz) increased expression of aggrecan, collagen type I and II, and decorin [ 234]. Aggrecan is a proteoglycan with primarily a mechanical function in the tissue matrix and is vital for disc health. Analysis of excised painful IVD has shown the presence of nerves and blood vessels that have intruded into the disc, and this has been suggested to be a result of altered aggrecan content in the IVD [ 235]. Decorin is a small proteoglycan produced by neurons and astrocytes and has an anti-inflammatory effect and regulates scarring in the spinal cord. For this reason, Decorin has been suggested as a treatment for spinal cord injury [ 236]. Biglycan is a small molecule that is important in the mineralization of bone and also plays a role in the osmotic swelling in the IVD, and versican is a large proteoglycan, which functions in cell adhesion and cell signaling [ 234]. Collagen type I and II are important components of the annulus fibrosus and nucleus pulposus respectively [ 234]. Taken together, the expression of these genes produces an anabolic effect, which improves the health of the discs and maintains its hydration and shock absorbing capabilities. In low back pain, abnormal mechanical loading leads to internal disc disruption [ 237, 238, 239], cell-mediated loss of water content and disc height, and is associated with a loss of aggrecan and collagen content within the disc [ 240, 241]. The disc releases proinflammatory cytokines promoting the infiltration and activation of immune cells that potentiate nerve and blood vessel growth further. The degenerated disc and the immune cells begin to produce neurogenic factors that activate nociceptive nerve signals to produce the sensation of disc related pain [ 242]. Therefore, vibration treatment may serve to counteract these types of effects seen in the progression of disc degeneration that leads to pain.

Application: Focused Vibration for Chronic Back Pain

5.1. Reporting Suggestions

An important observation from this review of vibration literature is how precisely the application of vibration is reported in studies. Research studies typically report some combination of the following: (1) the frequency of the vibratory stimulant in Hertz, (2) the amplitude in mm of the vibration, which is defined as the maximum displacement by a point on a vibrating body from its equilibrium position, (3) the acceleration of a point on the vibrating body measured in metric units (m/s ²) or units of gravitational constant g, where 1 g = 9.81 m/s ², (4) the pressure of vibration measured as dyn/cm ², which indicates the force of 1 g accelerated by 1 cm per second squared; or as pGz indicating periodic (p) vibrational acceleration (G) along the z axis (horizontal), which in research is typically between 1 and 4 Hz, and (5) the duration of the stimulant as well as how often it is applied over days/weeks/months.

There are two main inconsistencies regarding reporting the characteristics of vibrations. First, the inconsistency of what is considered "high frequency" and what is considered "low frequency". For example, some studies describe vibrations of 30-100 Hz as high frequency [ 206, 208, 220, 222] (the perspective of a whole body platform that moves mechanically), whereas other studies report anywhere from 0 to 200 Hz as low frequency [ 70, 115, 119, 155, 180] (the perspective of sound). This is largely due to the scientific contexts in which researchers are interested, but such contextual labels make the synthesis of knowledge in vibration more difficult than it needs to be. We recommend simply saying "vibration at X frequency". If one wishes to be descriptive, it is better to describe the nature of the vibration or applied area rather than saying it is high or low. For example, one may investigate the effects of percussive vibration, acoustic vibration, globally, or focally applied vibration. We used the term "low frequency" to describe 0-250 Hz vibration because this paper is presented in the context of music and sound.

The second inconsistency is in reporting of the amount of surface of the body that is stimulated and the relative distribution of the vibratory effect. For studies in WBV the entire body is stimulated but the actual vibratory transmission can be dependent on posture and stance. With vibroacoustic stimulation devices, the application can be almost the entire body with speakers above the knee, lower back, upper back, and neck [ 40] or primarily the back with a single transducer in the centre of the back [ 41], or only hands and feet with small piezoelectric devices [ 247], or as small as a pencil-like probe [ 32, 147]. Therefore, we recommend to add a sixth reporting category: (6) the region of the body that is directly stimulated by vibration (e.g., spine, low back, neck, feet, and whole-body).

As a result of the different methods of measurement, an important consideration is which measurements should be reported at a minimum to replicate studies of vibration on human health and accurately compare different studies. Not all six categories of measurement described above need to be reported because some allow conversion to other units. For example, displacement can be determined by velocity and frequency, velocity can be determined by frequency and displacement, acceleration can be determined by frequency and displacement, and pressure can be determined by force (a function of acceleration and mass) and the area applied. Therefore, reporting standards for vibration studies on health should have a pragmatic number of measurements that can allow conversions to other units if need be. We recommend a minimum of (4) measurements: the frequency (in Hz), the acceleration (in g), the duration of stimulus (time applied and over how many days), and the area of the body stimulated (e.g., spine, back, whole-body, or a measured area in units). The area of applied vibration is a new but important reporting standard to consider.

5.2. Clinical Implications

This attempt to "map the landscape" of the mechanisms of vibrational effect on the body has shown that research is beginning to identify how vibration affects "blood, brain, and bone" but that the means of the effect is highly complex. There is ample evidence that whether a person is standing in front of a tower of speakers at a rock concert, dancing to a pulsating beat, feeling the quake of pipe organ pedal tone, travelling on the underground train, riding a carriage on cobble stones, using a handheld vibrator, standing on an oscillating platform in a gym, sitting in a vibroacoustic chair, or having the spine treated with a vibrating stylus there is potential for effects that can affect health positively or negatively.

Throughout the paper we pointed to potential clinical applications. In some cases, these are already being used as in the case of whole body vibration for osteoporosis, or focal application to the spine for orthopedic conditions. However, most of the potential applications still need to be clinically recognized and commercialized in medically regulated products so that specific claims and, consequently, medical prescription can become generally practiced. The potential for this is indicated by the research in haemodynamics, in neurology, and in musculoskeletal conditions.

Professional organizations to promote and research these effects of vibration include VIBRAC ( https://www.vibrac.fi/) (accessed on 21 April 2021), which is focused on vibracoustic therapy for a large but mainly unspecified range of health conditions and WAVEX: World Association of Vibration Exercise Experts ( https://internationalwavexmeeting. wordpress.com/) (accessed on 21 April 2021) focused on WBV for physical and cognitive health.

Although mechanisms of action stemming from vibration appear to be clinically relevant in many situations, caution must be urged that in many cases the research employed animal studies. In many cases results from animal studies can be replicated in humans but that is not a certainty. Consequently, translation to clinical applications is not immediate and may not be successful.

5.3. Future Research

Our review has shown that much more specific research is needed. For example, research needs to determine whether vibroacoustic stimulation of the torso in a chair has similar effects as whole body vibration on a platform on the muscles in frail people. Research needs to explore more fully the effect of frequency on specific mechanisms. For example, how is nitric oxide positively correlated and prostaglandin E ₂ negatively correlated with frequency and, therefore, what is a target frequency for what specific effect. Similarly, our review showed examples of how vibration at one amplitude was positive and at another was negative. The complexity of mechanism response to vibration makes clinical application and conclusion of effect difficult. Interacting variables on multiple mechanisms including duration, strength, cycle, frequency, application location and area, and patient group make control of specific outcomes a challenge and frame the agenda for urgently needed research to advance this potentially potent form of treatment.

An important observation from this effort to "map the landscape" is that there is not an integrated "field" of vibrational research. Rather there are individual silos that seem to function in isolation of each other. This even applies to the type of stimulus. WBV has developed as one silo because of the device and strong application within athletics and sports training and medicine. Vibroacoustic therapy is another silo, less developed although not new, and with less awareness of mechanism in its research. The most diverse and so not even qualifying as a "silo" is focal vibration with applications ranging from orthopaedic treatment, to temporomandibular joint pain management, to orthodontic tooth movement, to tissue engineering, or to orthopedics. The reality may be that the person in the gym seeking to strengthen the muscles of the body core on a vibrating platform may be doing much more than they realize. It is time for these separate but related research streams to explore commonalities and shared research questions to move this clearly important field forward to advance clinical applications in healthcare.

AMA Style

Chicago/Turabian Style

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Possible Mechanisms for the Effects of Sound Vibration on Human Health

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This paper presents a narrative review of research literature to "map the landscape" of the mechanisms of the effect of sound vibration on humans including the physiological, neurological, and biochemical. It begins by narrowing music to sound and sound to vibration. The focus is on low frequency sound (up to 250 Hz) including infrasound (1-16 Hz).

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A future perspective for regenerative medicine: understanding the concept of vibrational medicine

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Regenerative Medicine has reached a new frontier, and understanding the concept of energy medicine and vibration science in relation to body physiology could provide us with a deeper understanding of the cellular processes that govern healing and perhaps unlock new avenues in tissue engineering. This paper is written with the intention of introducing vibration medicine as a new dimension to the expanding field of regenerative medicine.

In the expanding field of regenerative medicine, new avenues of research in the field of energy medicine and vibration science could unlock a deeper understanding of body healing mechanisms and the regeneration of tissue that could be applied for new therapies. This paper is written to influence research in the field of vibrational science in conjunction with tissue engineering.

In the rapidly expanding field of regenerative medicine, the focus is on replicating the inherent natural mechanisms in the body to perfection. The focus is on developing techniques that can mimic the perfectly engineered autoregulation's healing, cell division, cell maturation and homeostasis in tissue. We have come a long way in new avenues to replace damaged tissue, deliver stem cells in areas of degeneration, help heal skin wounds and reduce aging [ 1]. There is no doubt that the basis of creating perfection in the regenerative techniques is by a thorough understanding of the basic physiology of the human body and perhaps considerations beyond our current understanding of basic physiology. All theory brings us to question where the single cell programmed itself to become the entire organism, or the 'origin'. How close can we get to that origin, or the 'force' as most ancient texts refer to the energy that is the life force behind all processes in the body?

This leaves scientists with the interesting aspect of going back to understanding how this fascinating 'life force' can be harnessed and perhaps applied to the expanding field of regenerative medicine. This article seeks to inspire innovation in the field by summarizing what research has been performed in vibrational and energy healing in terms of regenerative medicine, and perhaps evolve them into newer applications of tissue engineering and regenerative therapies to reach the perfection we desire.

The concept of Biofield energy: mechanical & subtle energies in the evolution of vibrational medicine

The emerging science of the 'Biofield' aims to provide a scientific foundation for homodynamic regulation of living systems. By probing deeper into this field we arrive at an improved understanding of the foundations of biology and body physiology as well as the concept of Energy Medicine [ 2].

As per author and physician scientist Dr Richard Gerber [ 3], the Newtonian model of medical thinking sees the human physiological and psychological behavior as dependent upon structural hardware of the brain and body. However, the Newtonian mechanistic viewpoint of life is only an approximation of reality. In a machine, the function of the whole can be predicted by the sum of its parts; however, in reality, the human body is not a sum of its individual components; there is the force that 'animates.' Once that force leaves the body, as in after death, the body organs slowly deteriorate. A modern and more recent perspective of the life force has involved looking at it as a vibration science based on Einstein's paradigm. This concept sees human beings as a network of complex energy fields that interface with physical and cellular systems. Vibrational medicine uses specialized forms of energy to positively affect those energetic systems that may be out of balance due to diseased states [ 3].

Subtle energies are the foundation of the consideration of ethereal bodies [ 3], and not just the anatomical/physiological maps of what we consider in modern medicine. Ethereal bodies include maps and meridians of energy flow and chakra points. The lines of energy that can be mapped are called Nadis in Ayurveda and Qi energy lines in acupuncture. Over the decades, many forms of healing have looked at harnessing these subtle energies, such as Homeopathy, Reiki, reflexology, Yoga Therapy, acupuncture and Ayurveda. These are practiced today, and have a strong following of people that absolutely believe in these methods of healing [ 3].

From ancient medicine to the evolution of vibrational medicine

Various popular ancient medicine practices have utilized ethereal body maps to understand energy flow within the body

In the Chinese belief, the life force is the Qi energy (breath), also known as the 'Yang', which mixes with the blood, or the 'Yin energy', and creates the balance for life [ 4, 5]. Per this belief the imbalance of the Yin and the Yang creates the disease process. Qi travels throughout the body along 'Meridians', or special pathways. Acupuncture is based on the belief of inserting needles along certain energy points that would help improve and balance the Qi energy in proper flow [ 6, 7]. In this vein, one study reported that endomorphin-1, β endorphin, encephalin and serotonin levels increased in plasma and brain tissue through acupuncture application, which resulted in analgesia, sedation and recovery in motor functions [ 8].

Originating in India, Ayurveda is one of the ancient yet living health traditions [ 9]. Life in Ayurveda is conceived as the union of the body, senses, mind and spirit. Ayurveda is based on looking at the body as five elements that constitute the material component structure through which the prana (breath) energy flows through the 'Nadis' and animates the body. The five elements are water, earth, fire, ether and air [ 10]. The body is assessed on the basis of its Doshas; that is, expressed as Prakriti (phenotype), which broadly means a body type or individual nature. One clinical study showed a strong correlation between HLA alleles and Ayurvedic Prakriti type [ 11]. Later, it was also hypothesized that different Prakritis may possess different drug metabolism rates associated ayurvedic 'prakriti' types, suggesting a strong correlation to physiology and pathophysiology [ 12].

The practice of Yoga is said to use breath (Prana) energy to purify the body along with certain postures that have physiologic benefits to the body [ 13]. Several clinical trials based on assessing changes in the inflammatory markers in the body in response to the yogic lifestyle have shown improvement in overall health, and recovery from cancer and mental health issues [ 13-16]. This research demonstrates a clear link between these practices and health, and thus suggests their use might be widened to improve other areas of medicine, such as regenerative anti-aging.

Evidence-based literature looking at subtle energies, mind-body connection & electromagnetic fields

A New York-based orthopedic surgeon in his research reported the presence of electromagnetic waves around a healing tissue. Becker's [ 17] original work observed the phenomena known as the 'current of injury'. This is the electrical potential that can be measured from an amputated stump in an animal. When surgically removing a limb from an animal in the laboratory, the changes of the electrical patterns at the stump could be measured over several days during the healing and repair process [ 17]. More recently a paper by Hunckler et al. discussed how electrostimulation of wounds has been shown to be a promising treatment for accelerating regeneration in wounds [ 18]. Similarly, an insight into how the clock mechanisms of the stem cells in the skin respond to environmental cues in the shift in light energy are interesting insights into the energetic influences of the cell [ 19]. In the field of cosmetics, and antiaging of skin with the use of new-age laser devices, a photomechanical and photo thermal effect of the extracellular matrix of the dermis has been shown to cause neocollagenesis, thermal/photo-mechanical 'energy' that is an another example of the concept of vibration [ 20]. New ultrasound-based drug delivery systems use sound energy to deliver topical preparations deeper by influencing cellular ionic balance [ 21].

A recent study was designed to explore the impact of Yoga and Meditation based lifestyle intervention (YMLI) on cellular aging in apparently healthy individuals [ 22]. During this 12-week prospective, open-label, single arm exploratory study, 96 apparently healthy individuals were enrolled to receive YMLI. The primary end points were assessment of the change in levels of cardinal biomarkers of cellular aging in blood from baseline to week 12, which included DNA damage marker 8-hydroxy-2′-deoxyguanosine (8-OH2dG), oxidative stress markers reactive oxygen species and total antioxidant capacity, and telomere attrition markers telomere length and telomerase activity. The secondary end points were assessment of metabotrophic blood biomarkers associated with cellular aging, which included cortisol, β-endorphin, IL-6, BDNF and sirtuin-1. After 12 weeks of YMLI, there were significant improvements in both the cardinal biomarkers of cellular aging and the metabotrophic biomarkers influencing cellular aging compared with baseline values. The mean levels of 8-OH2dG, reactive oxygen species, cortisol and IL-6 were significantly lower and mean levels of total antioxidant capacity, telomerase activity, β-endorphin, BDNF and sirtuin-1 were significantly increased (all values) post YMLI. The mean level of telomere length was increased but the finding was not significant. YMLI significantly reduced the rate of cellular aging in apparently healthy population [ 22].

The healing power of thought is a concept that is looked at with clinical research done on meditation. A recent observation by scientist Hankey A has documented improvement in sensory acuity, perceptual style but also cognitive function, indicating stabilization of aspects of attentional awareness when comparing advanced practitioners of Tibetan Buddhist meditation in remote regions of the Himalayas, with established results on long-term practitioners of the Transcendental Meditation programs [ 23]. In another recent publication, the authors puts forth the hypothesis that mental phenomena are connected with thermodynamic properties of a large neural network [ 24].

In various cell migration studies, traction is a measurable unit, and traction measurements have been shown to be quantified. The 'force' of both traction and contraction can be measured [ 25]. The impacts of sound, heat and light on cells have been studied and documented, and cellular changes have been reported. If external forces have an impact on living cells, and we believe in the theory of equal and opposite reactions, there will be an internal force that is acting to negate the external force, and the balance of the two forces perhaps leads to cellular changes. Studies show specific electromagnetic field frequencies enhance human bone marrow stromal cell (hSSC/BMSC) adherence, proliferation, differentiation and viability, all of which play a key role in the use of hSSCs/BMSCs for tissue engineering in recreating cartilage tissue, bone and muscle [ 26]. For decades it has been reported that energy fields can play important roles in determining cell differentiation, migration, adhesion and even wound healing. Combinations of electric field, magnetic field and electromagnetic field techniques have revealed new and exciting explanations for how cells move and adhere to surfaces; how the migration of multiple cells are coordinated and regulated; and how cells interact with neighboring cells and to changes in their microenvironment [ 27].

Conclusion & future perspective: vibrational medicine & its future with regenerative medicine

As a scientist, one's journey involves unlocking secrets within the realm of the known body and cellular processes. We have only begun to understand pathways and cellular interfaces that have governed life for thousands of years. What lies beyond the concepts of known facts are the possible discoveries of unseen forces that regulate the many regulatory processes in the body. Learning from these ancient concepts of subtle energies brings to light possible explanations for cell behavior, autoregulation, tissue healing and understanding of forces beyond the reach of quantitate measurements as factors that play a role in regeneration and maintenance. As modern-day scientists, with modern equipment, can we take ancient concepts and utilize them?

The goal of this opinion article is to inspire more regenerative medicine research into the field of vibrational medicine, perhaps by looking to quantify the pressure used in acupuncture needles to create a proper flow of Qi for a patient to see a result [ 28], or measuring inflammatory markers after channeling Chakra healing and Chakra energy. Perhaps also to understand and teach the ethereal maps of the body as a mainstream course in medicine and basic science so that we can have a foundation knowledge of all possible ways of looking at the human body as a 'multidimensional being' [ 29]. We can then arrive at more modern applications of ancient knowledge that is current with our knowledge of the stem cells, wound healing and tissue engineering. There should be a meeting point where alternative healing and mainstream science merge and learn from each other. As Faculty, I applaud conferences that provide a platform for such merger, like the International Society of Regenerative Medicine [ 30]. The expansion in the regenerative medicine field will reach a point where controlling the internal forces and interactions between cells would be essential to perfect the healing technologies and perhaps slowing down the process of aging. But for that we must have a strong knowledge and foundation base of ancient techniques as scientists. The next wave of regenerative medicine scientist might be 'the spiritual scientist', focused on understanding energy flow in human physiology.

Introducing the concept of vibration science and energy mechanics in the body

Ancient practices that focus on body energy and vibrational healing.

Evidence based understanding and historical over view of vibration and the biofield to date.

Conclusion and future perspective: incorporating vibration medicine into regenerative medicine and tissue engineering.

Footnotes

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The author states that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Open access

This work is licensed under the Creative Commons Attribution 4.0 License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

References

Papers of special note have been highlighted as: * of interest; ** of considerable interest

7. Ponce Pedro E. Eastern medicine collides with Western regulations at mass. Acupuncture school. [ The Chronicle of Higher Education. 1993 Google Scholar]

11. Bhushan P, Kalpana J, Arvind C. Classification of human population based on [ HLA gene polymorphism and the concept of Prakriti in Ayurveda. [ PubMed] Google Scholar]

12. Ghodke Y, Joshi K, Patwardhan B. Traditional medicine to modern pharmacogenomics: ayurveda prakriti type and [[ CYP2C19 gene polymorphism associated with the metabolic variability. PMC free article] [ PubMed] Google Scholar]

24. Lefebvre VA. Theoretical modeling of the subject: Western and Eastern types of human reflexion. [ Prog. Biophys. Mol. Biol. 2017 Epub ahead of print. [ PubMed] Google Scholar]

30. International Society of Regenerative Medicine. 2017. www.isregen.org ISRegen.

Articles from Future Science OA are provided here courtesy of Future Science Group

# data[].attributes.attachment_link.title
A future perspective for regenerative medicine: understanding the concept of vibrational medicine

# data[].attributes.attachment_link.description
Regenerative Medicine has reached a new frontier, and understanding the concept of energy medicine and vibration science in relation to body physiology could provide us with a deeper understanding of the cellular processes that govern healing and perhaps unlock new avenues in tissue engineering.

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Therapeutic effects of whole-body vibration on fracture healing in ovariectomized rats: a systematic review and meta-analysis - PubMed

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Their conclusion, based on nine selected papers, is that Whole Body Vibration can promote bone healing, mineralization, and maturity and restore mechanical properties of bones.

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Affiliations

¹ Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China.

² Institute of Spine Disease, Shanghai University of Traditional Chinese Medicine, Shanghai, China.

³ School of Rehabilitation Science, Shanghai University of Traditional Chinese Medicine, Shanghai, China.

⁴ Key Laboratory of theory and therapy of muscles and bones, Ministry of Education, Shanghai, China.

Show details

Affiliations

¹ Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China.

² Institute of Spine Disease, Shanghai University of Traditional Chinese Medicine, Shanghai, China.

³ School of Rehabilitation Science, Shanghai University of Traditional Chinese Medicine, Shanghai, China.

⁴ Key Laboratory of theory and therapy of muscles and bones, Ministry of Education, Shanghai, China.

Abstract

Objective: Whole-body vibration (WBV), providing cyclic mechanical stimulation, has been used to accelerate fracture healing in preclinical studies. This study aimed to summarize and evaluate the effects of WBV on bone healing in ovariectomized rat models and then analyze its potential effects on fractures in human postmenopausal osteoporosis.

Methods: PubMed, EMBASE, Web of Science, China National Knowledge Infrastructure, VIP, SinoMed, and WanFang databases were searched from their inception date to September 2017, and an updated search was conducted in January 2018. Studies that evaluated the effects of WBV on bone healing compared with control groups in ovariectomized rats were included. Two authors selected studies, extracted data, and assessed the methodological quality. Meta-analyses were performed when the same outcomes were reported in two or more studies.

Results: Nine eligible studies were selected. In treatment groups, callus areas were significantly improved in the first 3 weeks, normalized total bone volume and total tissue volume values increased dramatically at 8 weeks, and the mechanical tests showed a significant difference at the end point of the study.

Conclusions: This study suggested that WBV could accelerate callus formation in the early phase of bone healing, promote callus mineralization and maturity in the later phase, and restore mechanical properties of bones.

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national center for biotechnology information

# data[].attributes.attachment_link.title
Therapeutic effects of whole-body vibration on fracture healing in ovariectomized rats: a systematic review and meta-analysis - PubMed

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1 Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, China. 2 Institute of Spine Disease, Shanghai University of Traditional Chinese Medicine, Shanghai, China. 3 School of Rehabilitation Science, Shanghai University of Traditional Chinese Medicine, Shanghai, China. 4 Key Laboratory of theory and therapy of muscles and bones, Ministry of Education, Shanghai, China.

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F1000Research Article: Vibration detection: its function and recent advances in medical applications.

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Medical Advances Through Vibration, Frequencies

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Medical Advances Through Vibration, Frequencies

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Oroszi T, van Heuvelen MJG, Nyakas C and van der Zee EA. Vibration detection: its function and recent advances in medical applications [version 1; peer review: 2 approved]. F1000Research 2020, 9(F1000 Faculty Rev):619 ( https://doi.org/10.12688/f1000research.22649.1)

NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.

Close Copy Citation Details

[version 1; peer review: 2 approved]

Tamás Oroszi Marieke J.G. van Heuvelen Csaba Nyakas 1,2, ³, ², Eddy A. van der Zee

https://orcid.org/0000-0002-6471-7938

¹

Tamás Oroszi Marieke J.G. van Heuvelen Csaba Nyakas 1,2, ³, ², Eddy A. van der Zee

https://orcid.org/0000-0002-6471-7938

¹

Author details Author details

¹ Molecular Neurobiology, Groningen Institute for Evolutionary Life Sciences (GELIFES), University of Groningen, Groningen, The Netherlands
² Research Center for Molecular Exercise Science, University of Physical Education, Budapest, Hungary
³ Department of Human Movement Sciences, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

Tamás Oroszi
Roles: Conceptualization, Writing - Original Draft Preparation, Writing - Review & Editing

Marieke J.G. van Heuvelen
Roles: Conceptualization, Writing - Review & Editing

Csaba Nyakas
Roles: Conceptualization, Writing - Review & Editing

Eddy A. van der Zee
Roles: Conceptualization, Writing - Original Draft Preparation, Writing - Review & Editing

Abstract

Vibrations are all around us. We can detect vibrations with sensitive skin mechanoreceptors, but our conscious awareness of the presence of vibrations is often limited. Nevertheless, vibrations play a role in our everyday life. Here, we briefly describe the function of vibration detection and how it can be used for medical applications by way of whole body vibration. Strong vibrations can be harmful, but milder vibrations can be beneficial, although to what extent and how large the clinical relevance is are still controversial. Whole body vibration can be applied via a vibrating platform, used in both animal and human research. Recent findings make clear that the mode of action is twofold: next to the rather well-known exercise (muscle) component, it also has a sensory (skin) component. Notably, the sensory (skin) component stimulating the brain has potential for several purposes including improvements in brain-related disorders. Combining these two components by selecting the optimal settings in whole body vibration has clear potential for medical applications. To realize this, the field needs more standardized and personalized protocols. It should tackle what could be considered the "Big Five" variables of whole body vibration designs: vibration amplitude, vibration frequency, method of application, session duration/frequency, and total intervention duration. Unraveling the underlying mechanisms by translational research can help to determine the optimal settings. Many systematic reviews on whole body vibration end with the conclusion that the findings are promising yet inconclusive. This is mainly because of the large variation in the "Big Five" settings between studies and incomplete reporting of methodological details hindering reproducibility. We are of the opinion that when (part of) these optimal settings are being realized, a much better estimate can be given about the true potential of whole body vibration as a medical application.

Keywords

Whole body vibration, cognition, translational research, Meissner corpuscles, Pacinian corpuscles, exercise, somatosensory, brain

Corresponding author: Eddy A. van der Zee Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2020 Oroszi T How to cite: Oroszi T, van Heuvelen MJG, Nyakas C and van der Zee EA. Vibration detection: its function and recent advances in medical applications [version 1; peer review: 2 approved]. First published: 17 Jun 2020, Latest published: 17 Jun 2020, 9(F1000 Faculty Rev):619 ( https://doi.org/10.12688/f1000research.22649.1) 9(F1000 Faculty Rev):619 ( https://doi.org/10.12688/f1000research.22649.1) F1000Research 2020, 9(F1000 Faculty Rev):619 ( https://doi.org/10.12688/f1000research.22649.1) et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Vibrations are oscillations that occur around an equilibrium point. They propagate in a certain medium (such as air, water, a branch of a tree, a leaf, or soil). The waveforms of the vibrations can range from very regular (sinusoidal) to very irregular (random). Such vibrations are all around us, produced, for example, by the wind, thunder in the air, or rain drops falling on the ground. They can also be caused by organisms of any size. Vibrations are often produced unintentionally while these creatures move around. Three basic forms of vibration transmission can be distinguished based on the medium in which the vibrations propagate: airborne, water-borne, and substrate-borne vibrations. These vibrations are all intricate components of the natural environment. It is assumed that they played an important role in the evolution of life1. Sensitivity to vibrations is found in even the simplest forms of life. Like the other senses, vibration detection is a crucial sense to be able to keep in touch with one's environment2. The vibration sense is used across the animal kingdom for various reasons: to detect prey, to avoid predators, to assess and navigate within a habitat or environment, or to search for food, amongst others. Vibrations are also used intentionally, for example to communicate with other individuals or to ward off a predator3,4. Also, humans detect vibrations, and even more so when other senses fail5. A present-day example is feeling a vibrating mobile phone in your hand or pocket. Also, sound is a form of vibration, which is collectively referred to as vibro-acoustics. A sound (or loud noise) can be both felt and heard by humans if both hearing sense and vibration sense are present. Given our sensitivity to vibration, it is somewhat surprising that we do not employ our capacity of vibration detection to the fullest. In this paper, we highlight one approach to employ vibrational sense: the use of whole body vibration (WBV) and its potential for medical applications. We summarize the latest advances in the use of WBV and stress that WBV has great potential as a therapeutic treatment once some critical issues are solved.

Vibration detection in humans

Humans are endowed with a high density of mechanoreceptors in the skin (notably in the fingertips and feet) to detect vibrations ( Figure 1). Next to those, mechanoreceptors can be found less abundantly in ligament, joints, blood vessels, and organs6. We evolutionarily inherited vibrational sensitivity, which is hard-wired in our body and brain. The mechanoreceptors project via the spinal cord and the thalamus to the somatosensory cortex. Various cortical brain regions are involved in vibrational information processing. Vibrations of high enough energy are therefore consciously detected. Vibrations of low energy or beyond the detection level of hearing (infrasound) still reach the brain, and it is believed that it causes annoyance and distress in many people7. Two main types of cutaneous mechanoreceptors receptive to vibrations are present in mammals, including humans (see 8 for review). Pacinian corpuscles, or pressure receptors, are deeply placed in the skin (and less abundantly elsewhere in the body)6. They sense vibrations at a range of 20-1,000 Hz, with a peak sensitivity around 250 Hz. Meissner corpuscles, or touch receptors, are more superficially placed in the skin. While touching a surface, the skin copies the surface via skin deformations and the corpuscles start to signal with their preferred frequency to the somatosensory cortex (see also Figure 2). Meissner corpuscles sense vibrations at a range of 5-150 Hz, with a peak sensitivity around 10-65 Hz. Meissner and Pacinian corpuscles are mandatory for the detection of vibrations. Stimulation of Meissner corpuscles results in the sensation of tapping-flutter-vibration, whereas stimulation of Pacinian corpuscles results in the sensation of vibration or tickling. Pacinian corpuscles in the human hand also serve a function in active texture exploration9. Under normal conditions, vibrational stimulation of the skin co-activates both types of skin mechanoreceptors either because the range of frequencies of the source is broad or because of harmonics (for example, a 30 Hz vibration will also generate vibrations of 60, 90, 120 Hz etc., although at a lower intensity; see for example10).

Recent advances in the use of vibrations: medical applications

Humans have a clear ability to detect vibrations of a wide range of frequencies and amplitudes. It had evident evolutionary advantages for individual survival (escaping predators or finding prey, and increased reproduction) for our ancestors. The use of the skin mechanoreceptors for sensing the texture of fruit for its edibility is considered a crucial step in our survival. These original advantages seem not so relevant anymore in our current society. However, vibrations and vibration detection are still linked to health issues. Numerous human studies on vibrations exist and are ongoing, examining under which conditions these vibrations become harmful. The aim of these studies is to determine which regulations are needed to prevent physical or mental damage. In general, vibrations can be dangerous for humans, notably when the amplitude is high (>1 millimeters), the duration is long (> ~30 minutes), and the vibrations have an erratic, random waveform. One aspect of harmful vibrations relates to the principle of resonance. Any part of the body has an intrinsic resonance frequency. The resonance frequencies will induce more physiological effects but if too strong should be considered harmful. For example, it has been estimated that the resonance frequency for the liver is 2-7 Hz in mice, while in humans the resonance frequency for the abdomen is 4-8 Hz, the thorax is 5-10 Hz, and the head is between 20 and 30 Hz11.

WBV research was in part inspired by determining these health risks. In contrast to harmful vibrations or harmful WBV, for example due to tools or driving heavy machinery, WBV can also be beneficial using vibrating platforms (see Figure 1 and Figure 2; "good" versus "bad" vibrations). The mechanical vibration of these vibrating platforms is transferred to the body, which triggers physical and physiological responses (see 12 for review). The type of vibration matters (vertical or side alternating and mechanical or sonic, for example) but also the subject's posture (sitting or standing on the plate in many different positions). An example of how this can affect the outcome is given by Alizadeh-Meghrazi and co-workers in individuals with spinal cord injuries13. Most often, the waveform is sinusoidal (and hence regular and predictable or predetermined). With a shift in focus from harmful vibrations to beneficial vibrations, the number of WBV publications increased (source: PubMed). Until around 2000, the number of WBV publications was about 15 per year. Then, the yearly number steadily increased and peaked in 2014 and 2015 (about 200 publications per year). The number of publications stabilized around 170 in later years. Systematic reviews started to appear around 2009 and reported on the effects of WBV on a wide variety of topics, including body composition, quality of life, pain management, musculoskeletal morphology and function, pulmonary functions, blood circulation and blood flow, many (neurological) diseases or conditions including musculoskeletal disorders, pulmonary function, chronic obstructive pulmonary disease, diabetes mellitus, fibromyalgia, osteoporosis, cancer, stroke, bone morphology, multiple scleroses, Parkinson's disease, Alzheimer's disease, cerebral palsy, and fracture healing. The often-recurrent conclusion of the systematic reviews is that findings are inconsistent, of rather modest effect size if present, and require further research. It signals a field in development with considerable potential but in need of efforts to pinpoint the true value of WBV as a therapy. Is the effect size of WBV interventions large enough to make it clinically relevant for many domains? Increasing awareness of the combined exercise and sensory component in WBV (explained in the next sections) can be viewed as a next step in an attempt to increase the effectiveness of WBV.

The exercise (muscle) component of WBV

WBV is used in sports as an exercise module. It can improve muscle functioning and bone structure (see 15 for review) in healthy patients as well as those with a medical condition16. Obviously existing WBV exclusion criteria for certain types of patients must be taken into account. WBV-exercise can be a stepping stone for more common forms of physical activity and exercise. Positive findings of WBV as exercise training on numerous outcome parameters keep appearing regularly. Providing a full overview of the recent developments in all of the exercise-related aspects of WBV research is, however, beyond the scope of the current review. In general, several new findings further stress that WBV is potentially a suitable rehabilitation training tool that warrants the continuation of investigations to increase reproducibility and effectiveness (see 17, 18, for example, and references therein). A recent review on WBV as a neuromuscular training method concluded that WBV can bring about improvements in muscle strength, power, and flexibility19. However, it depends not only on the characteristics of the WBV protocol and setting (mentioned above) but also on the characteristics of the participants. However, data are often insufficient to support or refute the use of WBV as an effective therapeutic intervention in diseased conditions. Despite the need for more consistency in WBV research, the steady stream of new publications has brought considerable new insights. Although an object of study since the 60s of the previous century, WBV is now becoming more clearly like a coin with two sides that can be further utilized if it comes to mode of action: the exercise component (e.g. muscles) and the sensory component (e.g. skin).

The sensory (skin) component of WBV

Obviously, these two components are not mutually exclusive. Skin signals and muscle signals (including proprioceptive signals not further addressed in this review) come together in the associative cortical areas of the brain. Traditionally, the exercise component is associated with WBV-exercise, training, and rehabilitation. However, the existence of the sensory component and the connection to the brain was already recognized in the early days of WBV research (see 12, for example). Focus of the field was still more on the potential discomfort and harmful aspect of WBV on the brain. It was linked to, for instance, motion sickness issues or the impact of vibrations in buildings negatively influencing quality of life20. The dopaminergic brain system becomes activated by WBV (20 Hz, 4G, for 90 minutes21), but by itself this is not necessarily bad for the brain. WBV also increased cerebral blood flow as was shown in a study in which low-frequency WBV protocols were used (ranging from 3-6 Hz)22. A review on peripheral blood flow reported that the change in it is frequency dependent23, and it can be assumed that higher peripheral blood flow also affects the brain. Choi and co-workers showed that 27 Hz WBV (but not 10 Hz WBV) increased brain activity in the prefrontal, motor, and somatosensory cortex24. That WBV can beneficially affect the brain is a rather recent insight. In a series of experiments, it was demonstrated that WBV (30 Hz vibrations) can improve cognitive performance in healthy young and older individuals, including individuals with attention deficits (ADHD)25-29. An fMRI imaging study in 2016 reported that after a session of WBV (by standing on a platform and providing a non-sinusoidal, random vibration of 7 Hz), the left caudate nucleus showed a significant increase in activity30. A more recent (pilot) study demonstrated the stimulating effects of WBV on the brain by examining electroencephalogram (EEG) activity. Alzheimer's patients in a relative early stage of the disease received WBV combined with varying standing positions on a platform (20 Hz at the start and every 2 weeks increased by 5 Hz, ending at 40 Hz). EEG activity increased significantly and was accompanied by a significant increase in cognitive performance of the participant31. Our own results with Alzheimer's patients at a much later stage of the disease (sitting on a WBV platform; see 32 for the experimental design) did not show improvements in cognitive performance. This may suggest that WBV has potential for Alzheimer's patients at an early stage of the disease only. Obviously, the many differences in the design of the study and the method to apply the vibrations make it difficult to generalize the impact of WBV on the brain. Nevertheless, finding an optimal setting (or settings) in which both components are fully utilized is a future goal in the WBV field.

The value of translational WBV research

Understanding the underlying (brain) mechanisms of WBV is a crucial step to optimize protocols. Which WBV settings are critical for a given study objective? Translational WBV research (positioning small rodents on a vibrating plate) provides a way to study the underlying mechanisms but only represent ~8% of publications. Of these studies, ~130 were done with rats and ~60 with mice as subjects. Recent advances in the translational approach revealed evidence for both the exercise and the sensory WBV components, but few investigations examined the brain. Ample data are present showing the impact of WBV on targets related to the exercise component of WBV. Providing such an overview is beyond the scope of this review. Only a limited number of (systematic) reviews are available on translational WBV research, but those available are positive about the therapeutic potential of WBV. One older study reviewed WBV data from humans, mice, and rats, and the authors considered WBV as a potentially therapeutic aid with what they called "global ramifications"33. A more recent study reviewed the WBV findings on fracture healing in ovariectomized rats34. Their conclusion, based on nine selected papers, is that WBV can promote bone healing, mineralization, and maturity and restore mechanical properties of bones. What about the sensory (skin) component in relation to the brain? Using cerebral ischemia in rats, researchers found that a 4-week WBV intervention regulated several brain markers in the cortical areas35. Mice subjected to 30 Hz WBV (low amplitude for 5 weeks; 10 minutes per day) showed an improvement in both memory and motor performance36. Brain analyses of mice following this type of WBV treatment revealed a significant increase in the activity of the cholinergic system37. A well-known role of the cholinergic system and its associated receptors is linked to attention38,39, and enhanced cholinergic activity is thought to contribute to the observed improvements in memory. Changed expression of neurotransmitters in the brain has also been found in an earlier study40 but in the context of long session duration (240 minutes). Long WBV sessions tend to eliminate positive effects on the brain36. A new insight is that the impact of WBV on the brain could also be mediated by the gut-brain axis. The role of the gut microbiome in regulating brain functioning is a booming field of research since the appearance of the seminal paper by Cryan and Dinan in 201241. The relevance of a healthy microbiome for general health in humans is now eminent. A recent review provides an overview of the evidence and potential underlying mechanisms42. So far, two papers appeared examining the impact of (sonic) WBV on the microbiome. Song et al. 43 demonstrated in both humans and mice that WBV affects the gut microbiome in terms of the diversity of the bacterial species present in certain areas of the gut. Yu and co-workers44 demonstrated a gut-microbial link between WBV treatment and the immune system in humans (both in the periphery and in the brain). The functional consequences of these findings cannot be determined easily owing to the complexity of both the gut microbiome and the gut-brain axis. WBV modulating the gut microbiome (possibly directly affecting the microbes) may nevertheless be an issue in future research related to microbial and inflammation-related disorders. In line with this, it was shown that WBV treatment could reduce the level of (neuro)inflammation in a stroke model using rats45.

The use of small rodents to understand human responses to WBV has also several limitations. For one, the rodents stand with four legs on the platform and cannot perform on command additional postures or combine it with exercise. They also possess vibration-sensitive whiskers used for tactile perception46, next to Meissner and Pacinian corpuscles in their paws. But a clear advantage is that they serve mankind as (genetic) models for many human diseases. Unravelling the underlying mechanisms of either beneficial or detrimental WBV effects remains of great value for human disease conditions and well-being.

Conclusion

It is clear that WBV is a research area that is expanding. It has clear potential for medical applications, but the field could benefit from more standardized and personalized protocols. The use of translational research can facilitate this need. The field should tackle what could be considered the "Big Five" of WBV: vibration amplitude, vibration frequency, method of application, session duration/frequency, and total intervention duration. One step forward will be an update of the guidelines for reporting WBV studies. These new guidelines are currently in progress by a team of WBV experts, preceded by a Delphi study47 in which 56 researchers are involved. In addition, knowledge of the underlying mechanisms should advance further. Once these steps have been made, a much better estimate can be given about the true potential of WBV as a therapeutic intervention and its value as a medical application.

Faculty Opinions recommended

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37. Heesterbeek M, Jentsch M, Roemers P, et al.: Whole body vibration enhances choline acetyltransferase-immunoreactivity in cortex and amygdale. J Neuro Transl Neurosci. 2017; 5(2): 1079. Reference Source

38. Schmitz TW, Duncan J: Normalization and the Cholinergic Microcircuit: A Unified Basis for Attention. Trends Cogn Sci. 2018; 22(5): 422-37. PubMed Abstract | Publisher Full Text | Faculty Opinions Recommendation

40. Ariizumi M, Okada A: Effects of whole body vibration on biogenic amines in rat brain. Br J Ind Med. 1985; 42(2): 133-6. PubMed Abstract | Publisher Full Text | Free Full Text

47. Wuestefeld A, Fuermaier ABM, van der Zee EA, et al.: Towards reporting guidelines of research using whole-body vibration in human subjects - A Delphi consensus study. PLoS One. 2020. in press

Author details Author details

¹ Molecular Neurobiology, Groningen Institute for Evolutionary Life Sciences (GELIFES), University of Groningen, Groningen, The Netherlands
² Research Center for Molecular Exercise Science, University of Physical Education, Budapest, Hungary
³ Department of Human Movement Sciences, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands

Tamás Oroszi
Roles: Conceptualization, Writing - Original Draft Preparation, Writing - Review & Editing

Marieke J.G. van Heuvelen
Roles: Conceptualization, Writing - Review & Editing

Csaba Nyakas
Roles: Conceptualization, Writing - Review & Editing

Eddy A. van der Zee
Roles: Conceptualization, Writing - Original Draft Preparation, Writing - Review & Editing

Grant information

The author(s) declared that no grants were involved in supporting this work.

Copyright

© 2020 Oroszi T et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Oroszi T, van Heuvelen MJG, Nyakas C and van der Zee EA. Vibration detection: its function and recent advances in medical applications [version 1; peer review: 2 approved] F1000Research 2020, 9(F1000 Faculty Rev):619 ( https://doi.org/10.12688/f1000research.22649.1)

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# data[].attributes.attachment_link.title
F1000Research Article: Vibration detection: its function and recent advances in medical applications.

# data[].attributes.attachment_link.description
Oroszi T, van Heuvelen MJG, Nyakas C and van der Zee EA. Vibration detection: its function and recent advances in medical applications [version 1; peer review: 2 approved]. F1000Research 2020, 9(F1000 Faculty Rev):619 ( https://doi.org/10.12688/f1000research.22649.1) NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article.

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A better way to control crystal vibrations: By introducing impurities to a material, researchers can control the speed and frequency of phonons, potentially leading to more energy-efficient devices.

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Fascinating Energy Efficiency Through Impurities

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Fascinating Energy Efficiency Through Impurities

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The vibrational motion of an atom in a crystal propagates to neighboring atoms, which leads to wavelike propagation of the vibrations throughout the crystal. The way in which these natural vibrations travel through the crystalline structure determine fundamental properties of the material. For example, these vibrations determine how well heat and electrons can traverse the material, and how the material interacts with light.

Now, researchers have shown that by swapping out just a small fraction of a material's atoms with atoms of a different element, they can control the speed and frequencies of these vibrations. This demonstration, published in Applied Physics Letters, by AIP Publishing, provides a potentially simpler and cheaper way to tune a material's properties, allowing for a wide range of new and more efficient devices, such as in solid-state lighting and electronics.

The natural vibrations of a crystalline material travel as particles called phonons. These phonons carry heat, scatter electrons, and affect electrons' interactions with light. Previously, researchers controlled phonons by dividing the material into smaller pieces whose boundaries can scatter the phonons, limiting their movement. More recently, researchers have engineered nanoscale structures, such as nanowires, into the material to manipulate phonons' speed and frequencies.

A team of researchers from the University of California, Riverside and the University of California, San Diego has now found that by doping -- introducing different elements into the material -- you can control phonons. The researchers doped aluminum oxide with neodymium, which replaces some of the aluminum atoms. Because neodymium is larger and more massive than aluminum, it alters the vibrational properties of the material, changing how phonons can travel.

"It introduces distortion to the lattice, which persists over a large distance compared to the atomic size, and affects the whole vibrational spectrum," said Alexander Balandin of the University of California, Riverside.

Using a new method of producing evenly doped crystals and new sensitive instruments to measure the phonon spectrum, the researchers showed, for the first time, that even a small number of certain dopants can have a big impact. "This approach provides a new way of tuning the vibrational spectrum of materials," Balandin said.

Previously, researchers assumed that any significant effect on phonons would require a very high concentration of dopants. But, the team found that doped aluminum oxide with a neodymium density of only 0.1 percent was enough to lower the phonon frequency by a few gigahertz and the speed by 600 meters per second.

Boosting phonon speeds increases a material's thermal conductivity, allowing tiny transistors to cool faster. Slowing phonons, on the other hand, would be useful in making more efficient thermoelectric devices, which convert electricity into heat and vice versa. Furthermore, in optical devices such as light-emitting diodes, slowing phonons and suppressing phonon interactions with electrons would mean more energy is used to produce photons (light) and less is lost as heat.

The researchers are now applying their strategy to other dopants and materials, such as gallium arsenide, with an eye toward developing energy-efficient devices, Balandin said.

# data[].attributes.attachment_link.title
A better way to control crystal vibrations: By introducing impurities to a material, researchers can control the speed and frequency of phonons, potentially leading to more energy-efficient devices.

# data[].attributes.attachment_link.description
The vibrational motion of an atom in a crystal propagates to neighboring atoms, which leads to wavelike propagation of the vibrations throughout the crystal. The way in which these natural vibrations travel through the crystalline structure determine fundamental properties of the material.

# data[].attributes.attachment_caption
The science behind healing crystals explained! - Times of India

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When one talks about the world of alternate therapies, the usage of healing crystals probably tops the list.
For the uninitiated, crystals (fossilized minerals) are believed to contain several healing properties. From bringing peace to the mind to fighting depression, the list seems to be endless. A lot of celebrities are using them and vouching for their efficacy. They have also been used in ancient forms of medicine and by priests to align the body chakras. And now, using crystals as an effective form of practicing self-care is becoming incredibly popular.
Scientifically, there is no science backing to the power of crystal healing in treating or curing conditions. However, using healing crystals can be very relaxing as they induce a sense of relaxation in the environment. But, do they actually work in treating medical conditions? We tell you
How do they work?

According to experts, crystals act as a power hold for healing as they allow positive, fruitful energy to flow into the body and do away with the negative, toxic energy. Like other forms of alternative therapy, crystals work by channelizing your energy levels, thereby, focus on healing your body from the inside.
Crystals also carry the power to induce a placebo effect in the body, which is scientifically proven to help medical treatment.
These healing rocks and crystals have their particular vibration and frequency, which arise from their molecular composition. From the way they move and interact, these vibrations and energies work in benefiting and uplifting our mood, mind, and health in a considerable manner, often in the way essential oils and aromatherapy work. Acting like a magnet, it can absorb the negative energies and welcome fresh vibes.
Since they are all naturally extracted, they harness the energies of the sun, moon, and oceans (all-natural healing energies) to improve our state. When you place or hold a crystal over the body, it interacts with the body's chakras and promotes physical and mental wellness. Used in a certain way, it can also improve your concentration and creativity. It can also promote physical, emotional and spiritual cleansing.
Crystals vibrate at the same pitch as humans and maximize the healing abilities we already have. Because of this comforting feeling, when you place a crystal on the body, it helps you connect better and feel at ease. Balancing crystals can also help match the misaligned energy levels.
How to use them? Which stone is the best?
Each healing crystal carries its own properties and powers. Depending on their benefits, you can use a healing rock to naturally heal yourself and remove stress and negativity. Some of the most commonly used stones include amethyst, rhodonite, opal, and rose quartz.
Amethyst has been found to hold powers which are extremely beneficial for the intestines and digestive troubles. Stones like green aventurine is good to promote cardiac wellness while healing rocks like yellow topaz clear up mental blocks and provide clarity.

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The science behind healing crystals explained! - Times of India

# data[].attributes.attachment_link.description
When one talks about the world of alternate therapies, the usage of healing crystals probably tops the list. For the uninitiated, crystals (fossilized minerals) are believed to contain several healing properties. From bringing peace to the mind to fighting depression, the list seems to be endless.

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Frequency In Medicine - We know it works - Three Dimensions Therapy College

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מכללת תרפיית מימדים

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Frequency In Medicine - We know it works - Three Dimensions Therapy College

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The Three-Dimensional Therapy College - We transfer extensive therapeutic knowledge for a better world. Learn more about Frequency In Medicine from our experts, Click Here!

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https://youtu.be/QjZhSxxhFgM

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Nikola Tesla Invented A Vibrational Healing Device Using This Frequency Code & It Actually Worked

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WARNING...Personally at 4.34 mins into video the machine that was being shown gave a noise which gave me sharp headache pain

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Nikola Tesla Invented A Vibrational Healing Device Using This Frequency Code & It Actually Worked

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Heal Any Disease: Physical Mental Spiritual Illness, Binaural Beats | 9 Solfeggio Frequencies

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WARNING this made me feel a bit nauseous and shaky with lower back pain after 6 minutes

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Heal Any Disease: Physical Mental Spiritual Illness, Binaural Beats | 9 Solfeggio Frequencies

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🧨Vibrational Medicine, Nik Tesla, EMF Debate⚡️ - Moneypenny Media NewsRoom

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🧨Vibrational Medicine, Nik Tesla, EMF Debate⚡️

w/ Moneypenny Nic, Carlos Boulden, AquaAmy 4H2O, Michael Tronic, Andy Jacobs, ( Mama Bear ), Michael Fedyk, Darrell Nichols - Been studying wavelengths, vibrations, radiationand the 'unseen' health benefits and dangers of tech innovation Now keen to share and debate Please bring ideas 💡

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🧨Vibrational Medicine, Nik Tesla, EMF Debate⚡️ - Moneypenny Media NewsRoom

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Monday, September 12 at 10:00pm BST with Moneypenny Nic, Carlos Boulden, AquaAmy 4H2O, Michael Tronic, Andy Jacobs, ( Mama Bear ), Michael Fedyk, Darrell Nichols. Been studying wavelengths, vibrations, radiationand the 'unseen' health benefits and dangers of tech innovation Now keen to share and debate Please bring ideas 💡

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Red Light Therapy Benefits - Does it Really Work?

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Red Light Therapy Benefits - Does it Really Work?

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How Piezoelectricity Works - Fusion 360 Blog

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Piezo what? Piezoelectricitysounds like a lot to take in, but it's simple to understand. The word piezoelectric originates from the Greek word piezein, which literally means to squeeze or press. Instead of squeezing grapes to make wine, we're squeezing crystals to make an electric current! Piezoelectricity is in a ton of everyday electronic devices, from quartz watches to speakers and microphones.

In a nutshell, piezoelectricity is the process of using crystals to convert mechanical energy into electrical energy or vice versa.

Regular crystals are defined by their organized and repeating structure of atoms that are held together by bonds, called a unit cell. Most crystals, such as iron, have a symmetrical unit cell. This makes them useless for piezoelectric purposes.

There are other crystals that get lumped together as piezoelectric materials. The structure in these crystals isn't symmetrical, but they still exist in an electrically neutral balance. However, if you apply mechanical pressure to a piezoelectric crystal, the structure deforms, atoms push around, and you have a crystal that conducts an electrical current. If you take the same piezoelectric crystal and apply an electric current to it, the crystal will expand and contract, converting electrical energy into mechanical energy.

Types of piezoelectric materials

There are a variety of piezoelectric materials that can conduct an electric current, both man-made and natural. The most well-known, and the first piezoelectric material used in electronic devices is the quartz crystal. Other naturally occurring piezoelectric materials include cane sugar, Rochelle salt, topaz, tourmaline, and even bone.

As piezoelectric technology started to take off after World War I, we began developing man-made materials to rival the performance of quartz. Man-made piezoelectric materials include:

PZT is from lead zirconate titanate and can produce more voltage than quartz with the same mechanical pressure.

Barium Titanate is a ceramic piezoelectric material discovered during World War II and is known for its long-lasting durability.

Lithium Niobate is a material that combines oxygen, lithium, and niobium together in a ceramic material that performs similarly to barium titanate.

How piezoelectricity works

We have specific materials suited for piezoelectricity applications, but how exactly does the process work? With the Piezoelectric Effect. The most unique trait of this effect is that it works two ways. You can apply mechanical or electrical energy to the same piezoelectric material and get the opposite result.

Applying mechanical energy to a crystal is a direct piezoelectric effect and works like this:

1. A piezoelectric crystal is placed between two metal plates. At this point, the material is in perfect balance and does not conduct an electric current.

2. Mechanical pressure is then applied to the material by the metal plates, which forces the electric charges within the crystal out of balance. Excess negative and positive charges appear on opposite sides of the crystal face.

3. The metal plate collects these charges, which can be used to produce a voltage and send an electrical current through a circuit.

That's it, a simple application of mechanical pressure, the squeezing of a crystal, and suddenly you have an electric current. You can also do the opposite, applying an electrical signal to a material as an inverse piezoelectric effect. It works like this:

1. In the same situation as the example above, we have a piezoelectric crystal between two metal plates. The crystal's structure is in perfect balance.

2. Electrical energy is then applied to the crystal, which shrinks and expands the crystal's structure.

3. As the crystal's structure expands and contracts, it converts the received electrical energy and releases mechanical energy in the form of a sound wave.

The inverse piezoelectric effect is used in a variety of applications. Take a speaker, for example, which applies a voltage to a piezoelectric ceramic, causing the material to vibrate the air as sound waves.

The discovery of piezoelectricity

Piezoelectricity was first discovered in 1880 by two brothers and French scientists, Jacques and Pierre Curie. While experimenting with various crystals, they discovered that applying mechanical pressure to specific crystals like quartz released an electrical charge. They called this the piezoelectric effect.

The next 30 years saw Piezoelectricity reserved largely for laboratory experiments and further refinement. In World War I, piezoelectricity was used for practical applications in sonar. Sonar works by connecting a voltage to a piezoelectric transmitter. This is the inverse piezoelectric effect in action, which converts electrical energy into mechanical sound waves.

The sound waves travel through the water until they hit an object. They then return back to a source receiver. This receiver uses the direct piezoelectric effect to convert sound waves into an electrical voltage, which a signal-processing device can then process. Using the time between when the signal left and when it returned, an object's distance can easily be calculated underwater.

With sonar a success, piezoelectricity gained the eager eyes of the military. World War II advanced the technology even further as researchers from the United States, Russia, and Japan worked to craft new man-made piezoelectric materials called ferroelectrics. This research led to two man-made materials used alongside natural quartz crystal, barium titanate, and lead zirconate titanate.

Piezoelectricity Today

In today's world of electronics, piezoelectricity is used everywhere. Asking Google for directions to a new restaurant uses piezoelectricity in the microphone. There's even a subway in Tokyo that uses the power of human footsteps to power piezoelectric structures in the ground. You'll also find piezoelectricity being used in these electronic applications:

Actuators

Actuators use piezoelectricity to power devices like knitting and braille machinery, video cameras, and smartphones. In this system, a metal plate and an actuator device sandwich together a piezoelectric material. Voltage is then applied to the piezoelectric material, which expands and contracts it. This movement causes the actuator to move as well.

Speakers & buzzers

Speakers use piezoelectricity to power devices like alarm clocks and other small mechanical devices that require high-quality audio capabilities. These systems take advantage of the inverse piezoelectric effect by converting an audio voltage signal into mechanical energy as sound waves.

Drivers

Drivers convert a low-voltage battery into a higher voltage which can then be used to drive a piezo device. This amplification process begins with an oscillator that outputs smaller sine waves. These sine waves are then amplified with a piezo amplifier.

Sensors

Sensors are used in various applications, such as microphones, amplified guitars, and medical imaging equipment. A piezoelectric microphone is used in these devices to detect pressure variations in sound waves, which can then be converted to an electrical signal for processing.

Power

One of the simplest applications for piezoelectricity is the electric cigarette lighter. Pressing the button of the lighter releases a spring-loaded hammer into a piezoelectric crystal. This produces an electrical current that crosses a spark gap to heat and ignite gas. This same piezoelectric power system is used in larger gas burners and oven ranges.

Motors

Piezoelectric crystals are perfect for applications that require precise accuracy, such as the movement of a motor. In these devices, the piezoelectric material receives an electric signal, which is then converted into mechanical energy to force a ceramic plate to move.

Piezoelectricity and the future

What does the future hold for piezoelectricity? The possibilities abound. One popular idea inventors are throwing around is using piezoelectricity for energy harvesting. Imagine having piezoelectric devices in your smartphone that could be activated from the simple movement of your body to keep them charged.

Thinking a bit bigger, you could also embed a piezoelectric system underneath highway pavement that the wheels of traveling cars can activate. This energy could then be used to light stoplights and other nearby devices. Couple that with a road filled with electric cars, and you'd find yourself in a net positive energy situation.

Want to help move piezoelectricity forward into the future? Get started with Fusion 360 Electronics today.

# data[].attributes.attachment_link.title
How Piezoelectricity Works - Fusion 360 Blog

# data[].attributes.attachment_link.description
Piezo what? Piezoelectricitysounds like a lot to take in, but it's simple to understand. The word piezoelectric originates from the Greek word piezein, which literally means to squeeze or press. Instead of squeezing grapes to make wine, we're squeezing crystals to make an electric current!

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The role of ATP signalling in response to mechanical stimulation studied in T24 cells using new microphysiological tools

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Abstract

The capacity to store urine and initiate voiding is a valued characteristic of the human urinary bladder. To maintain this feature, it is necessary that the bladder can sense when it is full and when it is time to void. The bladder has a specialized epithelium called urothelium that is believed to be important for its sensory function. It has been suggested that autocrine ATP signalling contributes to this sensory function of the urothelium. There is well‐established evidence that ATP is released via vesicular exocytosis as well as by pannexin hemichannels upon mechanical stimulation. However, there are still many details that need elucidation and therefore there is a need for the development of new tools to further explore this fascinating field. In this work, we use new microphysiological systems to study mechanostimulation at a cellular level: a mechanostimulation microchip and a silicone‐based cell stretcher. Using these tools, we show that ATP is released upon cell stretching and that extracellular ATP contributes to a major part of Ca ²⁺ signalling induced by stretching in T24 cells. These results contribute to the increasing body of evidence for ATP signalling as an important component for the sensory function of urothelial cells. This encourages the development of drugs targeting P2 receptors to relieve suffering from overactive bladder disorder and incontinence.

ATP, Ca Keywords: ²⁺ signalling, mechanostimulation, microphysiological systems, organ on chip, P2X, P2Y

1. INTRODUCTION

Dysfunction of the urinary bladder such as overactive bladder syndrome (OAB) is a common and stigmatizing problem. In OAB, the patient suffers from a disabling increase in micturition frequency and nocturia. 1 The prevalence of OAB is 11%‐19%, and the current options for pharmacological treatment are often associated with lack of efficiency or poor tolerability. 2 This condition impacts the quality of life of many patients worldwide, thus deserving further attention. Understanding the underlying molecular mechanisms is of outmost importance to help find new pharmacological treatments.

The current drugs to treat OAB are targeted towards cholinergic and adrenergic receptors; however, the contribution of purinergic nucleotides in bladder physiology was already described in 1972. 3 Purinergic mechanosensory transduction is a well‐known mechanism by which information about mechanical stress is relayed to the central nervous system via nerves expressing purinergic receptors. 4 This mechanism is of importance for urinary bladder physiology, which was clearly demonstrated in key studies using P2X3 knockout mice. 5, 6 The significance of P2X receptors and purinergic mechanosensory transduction in the urinary bladder was further established by Rong et al in a study describing P2X activation in low and high threshold afferent nerve fibres in the urinary bladders of mice. 7

A central component of purinergic mechanosensory transduction is the release of ATP from the affected cell or tissue as a result of mechanical stimulation. The release of ATP from urothelial cells upon mechanical stimulation has been described in a number of reports. 8, 9, 10 This has also been seen in erythrocytes as well as in endothelia. 11, 12, 13, 14 The current view of ATP in the voiding reflex can be summarized as follows: Distension of the urothelium triggers release of ATP which acts on P2X3 receptors on afferent sensory nerves in the suburothelium contributing to the sensation of bladder fullness. Efferent parasympathetic nerves innervate the detrusor smooth muscle. There, ATP is released as a co‐transmitter together with acetylcholine. ATP acts on P2X1 receptors on smooth muscle and contribute to the initiation of contraction. In the healthy human bladder, the purinergic contribution is only about 5% of the parasympathetic nerve‐mediated contraction, but this fraction is known to increase to about 40% in pathological bladder conditions. 15, 16, 17, 18 Of importance for the findings presented in this report are previous studies that have shown an increase in ATP in the urine from patients with OAB and bladder obstruction due to prostate hyperplasia. 19, 20

There are several comprehensive reviews available discussing the current knowledge of purinergic signalling in the urinary bladder. 21, 22, 23 Yet, purinergic signalling in the urinary bladder still has many aspects to be explored. For example, the urothelium itself, in addition to its ability to release ATP, also expresses both ionotropic P2X and metabotropic P2Y purinergic receptors. 24, 25, 26, 27, 28 This opens for the possibility that mechanical stimulation of the urothelium can initiate autocrine purinergic signalling, which in turn could contribute to cellular mechanotransduction. Cellular mechanotransduction, in this case, defined as the capacity of living cells to sense and integrate external mechanical stimuli and to convert them to intracellular signals. 29

In this study, we investigated the changes in intracellular Ca ²⁺ concentration induced in T24 urothelial cells by mechanical as well as direct ATP stimulation. We tested the hypothesis that ATP contributes to the Ca ²⁺ response induced by mechanical stimulation in T24 urothelial cells. To accomplish this, new microphysiological systems 30 for mechanical stimulation of cells in culture were introduced: a microfabricated chip, which allows mechanical stimulation and in situ recordings of the intracellular Ca ²⁺ concentration in single cells, as well as a silicon‐based stretch chamber for mechanical stimulation of a population of cells 31 (Figure 1 ).

2. MATERIALS AND METHODS

2.1. Cell culture

The bladder cancer cell line T24 (ATCC, no. HTB‐4) was propagated in Dulbecco's modified Eagle medium (DMEM) (Gibco) supplemented with 10% foetal bovine serum (FBS) (Gibco), 1% GlutaMAX (100X) (Gibco) and penicillin; streptomycin (100 U/mL; 100 mg/mL, Sigma‐Aldrich). Cells were detached from the cell‐culturing flask with 0.025%‐Trypsin‐EDTA (Gibco) in CaCl ₂/MgCl ₂‐free Dulbecco's phosphate‐buffered saline (DPBS) solution and washed once in DMEM, before 3 mL of cell suspension (1.0‐3.0 × 10 ⁵ cells/mL) was added to 30‐mm cell culture dishes (Sarstedt) with or without microactuator devices. Cells were incubated in a humidified 37°C, 5% CO ₂ cell incubator. The cell line was checked against the ICLAC Database of Cross‐Contaminated or Misidentified Cell Lines version 7.2. Cell culture was regularly screened for mycoplasma contamination by DNA staining using Hoechst 33258.

2.2. Calcium imaging

Loading of cells with Fura‐2 was performed during 40‐minutes incubation at room temperature in DMEM/F12 (Gibco) with 2 μmol L −1 Fura‐2‐AM (Life technologies) and 0.03% Pluronic F127 (Sigma‐Aldrich). Samples were mounted on a Nikon upright Eclipse 80i microscope with a Nikon Fluor 20X/0.5W dip down objective. Excitation at 340 and 380 nm was achieved with a DeltaRAM illuminator and a DeltaRAM‐V monochromator with a computer‐controlled SC‐500 shutter controller. Emissions (510 nm) were collected using a Photometrics Coolsnap CCD camera from Roper Scientific. Data were analysed with Image J (U. S. National Institutes of Health).

2.3. Mechanostimulation microchips

The microfabrication and operation of the mechanostimulation microchips have been described previously in more detail. 31 In short, on an oxidized Si wafer, an Au layer and a thin Cr adhesion layer were thermally evaporated. The photo‐patternable resin SU8 and electroactive polymer PPy were photolithographically patterned on the Au layer to form the different microactuators on the microchip (Figure 1 A). Next, the Au (and Cr) was wet chemically etched to form the final electrode structure, and the wafer was diced into single mechanostimulation microchips (Figure 1 B).

2.4. Cell stimulation

Cell stimulation experiments were performed at room temperature in DMEM without phenol red. Stock solutions of the different pharmacological agents were added to the cell culture dish comprising the cells to achieve the final concentrations. ATP, UTP, ADP, PPADS, apyrase (EC 3.6.1.5) and routine chemicals were acquired from Sigma‐Aldrich (St Louis, USA). Dilutions of stock solutions were prepared with deionized water (18.2 MΩ) before experiments. To test if the apyrase formulation had unspecific blocking effect not related to its enzymatic activity we tested if apyrase, which is a Ca ²⁺‐dependent enzyme, had any blocking effect in Ca ²⁺ ‐free media.

To perform mechanical stimulation, the microchip comprising the cells was mounted in a customized chamber in DMEM/F12 without phenol red. The mechanostimulation microchips were operated using a Gamry potentiostat Ref600 with Gamry PHE200 software. For Ca ²⁺ imaging, a 300 seconds stimulation of −1.0 V was followed by a period of 60 seconds at 0.0 V against the Ag/AgCl reference electrode. More details about the stimulation can be found in. 31

2.5. Construction of silicon‐based cell stretch chamber

Cell culture chambers, were fabricated by moulding silicon elastomer in an aluminium master form. Sylgard 184 Elastomer kit from DOW CORNING (USA) was used. Ratio of curing agent and base was 1:10. After pouring the mix into the master form, it was left in room temp until degassed and then cured in an oven at 70°C for 24 hours. The cured cell stretch chambers were cleaned by incubation in 70% ethanol and then in distilled water. Before use, they were autoclaved in 120°C for 20 minutes. The chamber was seated on a pneumatic system driven by a syringe pump as shown in Figure 1 C. Infusing the syringe with a certain volume controls the degree of stretch applied to the chamber. We used an infusion of 1 mL to accomplish a degree of cell stretch that corresponded to a dislocation of approximately 20%.

2.6. Measurement of ATP and LDH release

In the series of stretch‐induced ATP release experiments, T24 cells were cultured in PDMS cell culture chambers coated with 2 μg/cm ² laminin (Sigma‐Aldrich) in the same condition as mentioned above. Samples were taken and placed on ice immediately before and after stretching. A luciferin‐luciferase‐based reaction kit (Molecular Probes) was used to analyse extracellular ATP release. As control samples, cell lysate was prepared by lysing T24 cells cultured in the stretch culture chambers by adding 0.1% Triton X‐100 in PBS for 10 minutes at room temperature. Samples were analysed in 96‐well plates. LDH release was also analysed using Pierce™ LDH Cytotoxicity Assay Kit acquired from ThermoFisher Scientific. Analysis was performed in 96‐well plates according to the suppliers' instructions. ATP and LDH samples were analysed using a Spectra Max 190 plate reader (Molecular Devices).

2.7. Immunochemistry

For immunocytochemistry experiments, cells were cultured on coverslips. Prior to staining, cells were fixed in 4% formaldehyde for 10 minutes and then rinsed in PBS. Specimens were pre‐incubated in PBS containing 0.05% Triton X‐100 and 0.2% bovine serum albumin (BSA; both from Sigma‐Aldrich, St Louis, USA). Slides were then incubated with primary antibodies; guinea pig anti‐P2X3 (1:250; Novus Cat# NB100‐1658, RRID:AB_10001676, rabbit anti‐P2Y ₆ (1:50; H‐70; Santa Cruz Biotechnology Cat# sc‐20127, RRID: AB_2156250), diluted in PBS containing 0.05% Triton X‐100 and 0.2% BSA for 1 hour in a moist chamber at room temperature. After washing in PBS, the slides were incubated with secondary antibodies: goat anti‐rabbit antibody labelled with Cy 3 (1:500; Jackson ImmunoResearch Labs Cat# 111‐165‐144, RRID:AB_2338006) or goat anti‐guinea pig antibody labelled with Cy5 (1:500; Novus Cat# NB120‐6567, RRID:AB_790417), actin‐binding FITC‐phalloidin (1 μg/mL; Sigma‐Aldrich) and Hoechst 33258 (1:2000; Sigma‐Aldrich, 94403) for one hour at room temperature in a moist chamber. Slides were then rinsed in PBS before mounted with Dako Fluorescence Medium (DakoCytomation, Glostrup, Denmark) to prevent fading. Secondary antibody specificity was assessed by omitting the primary antibody. Human bladder tissue was used as positive controls. Collection of human bladder tissue was carried out in accordance with the Declaration of Helsinki and was approved by the Regional Ethics Committee at Karolinska University Hospital (Dnr. 2010/574‐32, 2009/1481‐32, 2008/1633‐31). All collected tissue was prepared as previously described. 28 Immunofluorescence image acquisition was performed with a laser scanning confocal microscope Zeiss LSM 800. Image analysis was conducted using Image J (U. S. National Institutes of Health).

2.8. Statistics and data analysis

All data are presented as mean ± SEM. Student's paired and unpaired t tests were used for comparison of differences between two groups, in cases where the data were not normally distributed according to Shapiro‐Wilk test, Mann‐Whitney U test was used instead of t test. For comparisons between more than two groups, ANOVA with Bonferroni correction for multiple comparisons was used.

3. RESULTS

3.1. Changes in cytosolic Ca²⁺ concentration by mechanical stimulation and ATP

T24 cells were cultured on mechanostimulation microchips based on the electromechanically active polymer polypyrrole (PPy) illustrated in Figure 1 A,B. The mechanochip delivers a vertical stretch of approximately 1 μm to cells cultured on the active sites of the chip. 31 When mechanical stimulation was applied to T24 cells, they responded with an increase in the intracellular Ca ²⁺ concentration as can be seen from the lower tracing in Figure 2 A. Cells cultured on the control PPy surface received the same electric stimulation (−1 V) and responded with peaks in cytosolic Ca ²⁺ concentration coinciding with the changes of the electrical current. Next, ATP was used to stimulate the T24 cells. Accumulative application of ATP from 10 −6 to 10 −4 mol L −1 resulted in a similar increase in the intracellular Ca ²⁺ concentration as seen with mechanical stimulation, as shown in Figure 2 B. The amplitude of cytosolic Ca ²⁺ increase seen with the mechanical stimulation corresponded to the effects of exogenous application of 10 −5 mol L −1 ATP (Figure 2 C).

To investigate if the mechanisms that lead to the increase in intracellular Ca⁺² concentration seen with both mechanical stimulation and ATP are connected, we inhibited the ATP‐induced cytosolic Ca ²⁺ increase, using the ATP‐diphosphohydrolase apyrase. As shown in Figure 3 , 10 U/mL apyrase blocked 90% of the increase in intracellular Ca ²⁺ induced by 10 −4 mol L −1 ATP. When mechanical stimulation was administered to cells on mechanochips, 10 U/mL apyrase blocked increase in cytosolic Ca ²⁺ by more than 65%. The blocking effect of apyrase was significant for both ATP stimulation as well as mechanical stimulation.

3.2. ATP release induced stretching of cells

The blocking effect of apyrase on the increase in intracellular Ca ²⁺ induced by mechanical stimulation suggests that release of ATP upon mechanical stimulation contributes to this response. To investigate if ATP is also released upon mechanical stimulation of a population of cells, we developed a stretch device (Figure 1 C) for stretching of an entire population of cells as the mechanochip only activates single cells located on the active sites. Stretching of cells at a dislocation of approximately 20% (Figure 4 A) resulted in a two‐ to threefold increase of the ATP concentration in the cell culture media as shown in Figure 4 B. Cell damage due to stretching defined by lactate dehydrogenase release was 1.1% ± 0.3% (mean ± SEM, n = 5).

3.3. Characterization of ATP response

To further understand the signalling pathways induced by stretching, the purinergic receptors involved in the ATP‐induced increase in cytosolic Ca ²⁺ concentration were investigated. Receptors of the P2X family are ligand‐gated ion channels, and therefore, the increase in intracellular Ca ²⁺ concentration mediated by the P2X receptors is dependent on extracellular Ca ²⁺. P2Y receptors, on the other hand, are G‐protein‐coupled receptors that can induce release of Ca ²⁺ from intracellular stores via the IP3 pathway. 32 To differentiate between P2Y and P2X, we stimulated T24 cells with ATP in media containing Ca ²⁺ as well as in Ca ²⁺‐free media. ATP dose‐response curves from 10 −6 to 10 −4 mol L −1 were generated, and the cells responded with a dose‐dependent increase in intracellular Ca ²⁺ regardless if there were Ca ²⁺ in the media or not (Figure 5 A). However, the response was lower in the Ca ²⁺‐free media; this difference was statistically significant. In Ca ²⁺ ‐free media, addition of apyrase did not have a blocking effect.

The effect of the non‐selective purinergic blocker PPADS was tested in T24 cells (Figure 5 B). PPADS at concentrations of 10 −6 mol L −1 and 10 −5 mol L −1 gave a statistically significant decrease in the intracellular Ca ²⁺ response to ATP.

P2Y receptor agonists, UTP and ADP, were tested, and dose‐response curves from 10 −6 to 10 −4 mol L −1 were generated. Addition of UTP as well as ADP led to an increase in the cytosolic Ca ²⁺ concentration, which was slightly less than that seen with ATP (Figure 5 C). Compared with purinergic receptors, agonists like carbachol, serotonin and histamine barely affected intracellular Ca ²⁺ concentration in T24 cells up to concentrations of 10 −4 mol L −1 (Figures S1 and S2).

3.4. Expression of P2 receptors in T24 cells

Polyclonal antibodies against the G‐protein‐coupled receptor P2Y ₆ as well as the ion channel P2X3 were used to identify these receptors on the T24 cells using immunocytochemistry. These receptors were chosen because they have been shown to be present in urothelial cells. 26, 28, 33 Immunoreactivity was seen with antibodies against both P2 receptors, see Figure 6 . Hoechst 33258 was used to visualize the cell nucleus, and the actin‐binding toxin phalloidin conjugated to FITC was used to stain actin to delineate the contours of the cells. Immunoreactivity was present throughout most of the cell with the most intense staining just outside the nucleus. Indicating a significant part of the immunoreactivity may be in the cytoplasm. In negative controls where the primary antibody was omitted, no immunoreactivity was seen (data not shown). Human bladder tissue was used as positive controls (Figure S3).

4. DISCUSSION

In this study, we set out to test the hypothesis that ATP is important for the changes in cytosolic Ca ²⁺ concentration induced by mechanical stimulation of T24 urothelial cells. To accomplish this, we introduced new tools, a mechanostimulation microchip and a silicon‐based cell stretcher, which we believe are of value for anyone interested in cellular mechanotransduction. We show that ATP is released from T24 cells upon mechanical stimulation; by blocking extracellular ATP, we could see a significant decrease in the response induced by mechanical stimulation. In addition, we showed that T24 cells express different subtypes of purinergic receptors, both from the families of ionotropic P2X and metabotropic P2Y.

Many studies have shown that ATP is released from urothelial cells upon mechanical stimulation. 10, 34, 35 Several reports also stress the importance of ATP as a non‐adrenergic, non‐cholinergic transmitter for signalling between the bladder and the central nervous system. 5, 36, 37, 38 This crosstalk where information about mechanical stimulation is relayed by purinergic signalling of sensory and parasympathetic nerves is well described in terms of mechanosensory transduction. The focus of this paper has been limited towards the role of auto‐ and paracrine purinergic signalling as a mechanism involved in cellular mechanotransduction. The exact role of ATP in cellular mechanotransduction in the urinary bladder is unclear. Some reports show an increase in intracellular Ca ²⁺ concentration before release of ATP, indicating that the mechanosensing is initiated by mechanosensitive, possibly Ca ²⁺‐conducting receptors. 39, 40, 41 These findings suggest that the release of ATP is an active secretion that requires an increase in intracellular Ca ²⁺ concentration to take place. Several mechanisms have been proposed for the release of ATP, for example electrodiffusional translocation via connexin‐ and pannexin‐containing hemichannels and voltage gated‐dependent anion channels, as well as facilitated diffusion by nucleotide‐specific ATP‐binding cassette (ABC) transporters and vesicle‐mediated release. 42 For urothelial cells, there is compelling evidence that hemichannels is the more important. 25, 26

Based on the results from this study, we suggest that the release of extracellular ATP and subsequent auto‐ and paracrine signalling are important factors for the initiation of a change in the cytosolic Ca ²⁺ concentration due to mechanical stimulation in T24 cells.

The increase in intracellular Ca ²⁺ concentration that we observe upon mechanical stretching is of the same magnitude as that obtained by adding 10 −5 mol L −1 of ATP. This is in line with other reports where concentrations of 10 −4‐10 −5 mol L −1 of ATP were used to induce changes in cytosolic Ca ²⁺ concentration in cells. 41, 43 The mechanical stimulation is accomplished with an electrically driven mechanochip. The chip contains control areas where cells are exposed to the potential differences and currents that drive the actuators, but without the mechanical stimulation. The cells on the control areas show fluctuations in the intracellular Ca ²⁺ concentration that coincide with the ion fluxes associated with the potential changes. We have seen these fluctuations previously and are currently investigating the exact nature of this phenomenon. By comparing the response of the stretched cells with the non‐stretched but electrochemically addressed cells, we can subtract the latter effect. Also, the mechanical expansion profile (expansion vs time) of the actuators is congruent with the response of the stretched cells, thus further indicating that the response is mechanically induced. The response we see with the mechanochip is long‐lasting and continues past the point where the potential is returned to zero. We think this lasting response is due to the fact that the PPy remains expanded, that is the strain on the cells remains even after the potential is returned to zero. We are currently developing a second generation of mechanochips where the strain will be reversible so that we can investigate the whole cycle of stretch and release on the cells. More detailed technical specifications of the mechanochip can be found in Svennersten et al 31

Furthermore, we observed that we could significantly decrease the response induced by either ATP or mechanical stimulation, using apyrase. However, the blocking effect was stronger for ATP‐induced stimulation than for mechanically induced stimulation. This could be due to the fact that urothelial cells express many other mechanosensitive receptors that may contribute to the increase in intracellular Ca ²⁺ concentration and that the autocrine ATP signalling works rather like an amplifier for the increase in the cytosolic Ca ²⁺ concentration. 44

We suggested that the significant blocking of the response induced by mechanical stimulation using apyrase suggested that the mechanical stimulation indeed resulted in release of ATP and subsequent autocrine signalling. To corroborate this finding, we measured ATP release upon mechanical stretching of the cells. Because the mechanochips only stimulate single cells, the released quantities of ATP from a single cell were too small to be measured using the luciferin‐luciferase assay. Therefore, we devised an alternative stretching tool using silicone elastomer that could stretch a whole population of cells, thus ensuring the release of a measureable amount of ATP. We observed that this stretching resulted in a significant release of ATP, which is in accordance to what others have found in bladder tissue experiments. 39, 40

As there are two major families of ATP receptors, the ionotropic P2X and the metabotropic P2Y, we wanted to investigate if both of these contributed to the response that we measured or whether one family of receptor was more dominant than the other. Our first approach was to perform the experiments in Ca ²⁺‐free media, as P2X receptors depend on extracellular Ca ²⁺. The responses seen in Ca ²⁺‐free media were slightly smaller than those seen in media containing Ca ²⁺, this difference was statistically significant. Upon testing agonists such as ADP and UTP, which activate P2Y rather than P2X, responses were slightly smaller compared to ATP. This finding suggests that both P2Y and P2X receptors are expressed by T24 urothelial cells and that they have a significant contribution to the Ca ²⁺ response seen upon stimulation with ATP. The role of P2Y receptors is important as a significant part of purinergic auto‐ and paracrine signalling in the bladder is composed of the metabolite ADP likely due to NTPDases expressed in the urothelium. 34, 45, 46, 47 We also tested other pharmacological substances such as carbachol, serotonin and histamine. The effect of these substances was barely measurable. In the case of carbachol, this is interesting since activation of nicotinic as well as muscarinic acetylcholine receptors has been shown to trigger release of ATP from urothelial cells. 48, 49 However, those studies did not use carbachol. Sui et al showed that carbachol can trigger release of ATP from urothelial cells but without significant increase in intracellular Ca ²⁺. 50 Therefore, it would be interesting in future studies to test if carbachol can trigger Ca ²⁺ ‐independent ATP release in T24 cells as well.

From the former findings, we conclude that P2Y receptors make a major contribution to the observed response. To further verify this, we tested the non‐selective purinergic receptor blocker PPADS, which is primarily described as a P2X antagonist at moderate concentrations. PPADS had a partial inhibitory effect, strengthening the conclusion that the observed responses comprised a large component of P2Y activity. However, some caution should be taken in making conclusions based on the PPADS results, as PPADS is reported to antagonize both P2X receptors at moderate concentrations 51 and P2Y receptors at high concentrations. 52 We saw relatively large blocking effect using PPADS at moderate concentrations compared to the effect of using Ca ²⁺‐free media, indicating that even at the concentration of PPADS that we use, there is some effect also on P2Y receptors. PPADS also has dual actions not only as an antagonist of P2‐purinoceptor but also as an ecto‐ATPase inhibitor. 53

With compelling pharmacological evidence for P2 receptor signalling, we wanted to investigate the expression of P2 receptors using immunochemistry methods. Previous studies had shown no effect on cAMP upon activation of P2Y in T24 cells making it likely that P2Y receptors in T24 cells are G q‐coupled rather than G s/i‐coupled. 54 Therefore, we chose to look for the expression of a P2Y receptor that was known to be G q‐coupled and also known to be found in urothelial cells, such as P2Y ₆. 32 We also investigated the expression of P2X3 receptors based on previous immunohistological findings from the human urothelium. 28 We found that in the T24 cells, there was immunoreactivity for both P2Y ₆ and P2X3, which is not surprising as these are previously shown in urothelial cells. 24, 26, 28, 33 However, regarding P2Y ₆ our functional data neither support nor oppose the presence of this subtype as it is mainly activated by UDP, only to a lesser extent by UTP and barely by ADP and ATP. 32 With this in mind, we think that it is very likely that also other subtypes are present. The pattern of the immunoreactivity for P2X3 and P2Y ₆ seen in this paper indicates that a significant part of receptors is located intracellularly; this might indicate that the cells have a continuous overexpression of these receptors which would not be surprising as the cells are derived from a urothelial cancer. Worth mentioning is that the distribution of immunoreactivity is similar to that of others who have stained P2Y receptors in cell cultures. 55 As a counterstain, we chose to use actin‐binding phalloidin rather than antibodies against cytokeratins. The main reason for this was to avoid possible cross‐reaction that might occur when using several antibodies simultaneously. Also staining of actin serves the purpose well to give clear delineation of the cells borders.

The work conducted in this paper is based on the T24 urothelial cell line, which is isolated from an urothelial carcinoma. This is a major limitation, and one should be careful not to extrapolate our results to mechanisms present in the healthy urinary bladder. However, the T24 is a robust cell line suitable for conducting this study where we introduce new tools to study mechanotransduction on a cellular level. In this work, we have employed new microphysiological systems for mechanical stimulation, a mechanostimulation microchip and a silicon‐based cell stretcher, and established basic principles of mechanotransduction on a cellular level. Our next step is to apply these methods and to test our hypothesis on human primary cells and tissues. We also hope that these new tools might serve as inspiration for our peers in the field of mechanotransduction.

Conclusively, T24 cells express P2Y and P2X receptors, both of which contribute to the change in cytosolic Ca ²⁺ concentration seen upon mechanical stimulation. However, P2Y seems to be the dominating contributor to this response. The autocrine ATP signalling associated with mechanotransduction in urothelial cells contributes to a major part of the mechanotransduction‐mediated changes in cytosolic Ca ²⁺ concentration in T24 cells. The mechanostimulation microchips and silicon‐based cell stretcher are effective, new tools to study mechanostimulation pathways at the cellular level.

AUTHOR CONTRIBUTION

NNG, KS and EWHJ conceived and designed the study. NNG and KS performed calcium and stretch experiments. EWHJ designed and fabricated microchips. NS and KS performed immunochemistry experiments. NNG, NS, EWHJ and KS prepared figures. NNG, EWHJ and KS wrote the first draft of the manuscript. KHG contributed control tissue and evaluated immunohistochemistry data. All authors contributed with editing of the paper and approved the final version.

CONFLICT OF INTEREST

The authors declare that they have no competing interests.

ACKNOWLEDGEMENTS

This study was supported by the Swedish Research Council (VR‐2014‐3079), the Lars Hiertas Minne Foundation, Ollie and Elof Ericssons Foundation for Scientific Research, Linköping University and Karolinska Institutet. Special thanks to Agneta Richter‐Dahlfors and her staff at the Swedish Medical Nanoscience Centre for generous access to Ca ²⁺ imaging facilities. Special thanks also to Professor Lars Gustafsson for kind mentorship and fruitful discussions and to Jenny Archer for language editing. Thanks to Professor Stefan Eriksson and the Department of Physiology and Pharmacology, Karolinska Institutet for support. Thanks to Professor Peter Wiklund at Department of Urology, Karolinska University Hospital for support. Thanks to the CLICK imaging facility founded by Knut and Alice Wallenberg Foundation.

REFERENCES

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The role of ATP signalling in response to mechanical stimulation studied in T24 cells using new microphysiological tools

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The capacity to store urine and initiate voiding is a valued characteristic of the human urinary bladder. To maintain this feature, it is necessary that the bladder can sense when it is full and when it is time to void. The bladder has a specialized epithelium ...

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Most materials of interest currently being investigated for photothermal therapy are on the nanoscale. One of the key reasons behind this is the enhanced permeability and retention effect observed with particles in a certain size range (typically 20 - 300 nm). Molecules in this range have been observed to preferentially accumulate in tumor tissue. When a tumor forms, it requires new blood vessels in order to fuel its growth; these new blood vessels in/near tumors have different properties as compared to regular blood vessels, such as poor lymphatic drainage and a disorganized, leaky vasculature. These factors lead to a significantly higher concentration of certain particles in a tumor as compared to the rest of the body.

Huang et al. investigated the feasibility of using gold nanorods for both cancer cell imaging as well as photothermal therapy. The authors conjugated antibodies (anti-EGFR monoclonal antibodies) to the surface of gold nanorods, allowing the gold nanorods to bind specifically to certain malignant cancer cells (HSC and HOC malignant cells). After incubating the cells with the gold nanorods, an 800 nm Ti:sapphire laser was used to irradiate the cells at varying powers. The authors reported successful destruction of the malignant cancer cells, while nonmalignant cells were unharmed.

When AuNRs are exposed to NIR light, the oscillating electromagnetic field of the light causes the free electrons of the AuNR to collectively coherently oscillate. Changing the size and shape of AuNRs changes the wavelength that gets absorbed. A desired wavelength would be between 700-1000 nm because biological tissue is optically transparent at these wavelengths. While all AuNP are sensitive to change in their shape and size, Au nanorods properties are extremely sensitive to any change in any of their dimensions regarding their length and width or their aspect ratio. When light is shone on a metal NP, the NP forms a dipole oscillation along the direction of the electric field. When the oscillation reaches its maximum, this frequency is called the surface plasmon resonance (SPR). AuNR have two SPR spectrum bands: one in the NIR region caused by its longitudinal oscillation which tends to be stronger with a longer wavelength and one in the visible region caused by the transverse electronic oscillation which tends to be weaker with a shorter wavelength. The SPR characteristics account for the increase in light absorption for the particle. As the AuNR aspect ratio increases, the absorption wavelength is redshifted and light scattering efficiency is increased. The electrons excited by the NIR lose energy quickly after absorption via electron-electron collisions, and as these electrons relax back down, the energy is released as a phonon that then heats the environment of the AuNP which in cancer treatments would be the cancerous cells. This process is observed when a laser has a continuous wave onto the AuNP. Pulsed laser light beams generally results in the AuNP melting or ablation of the particle. Continuous wave lasers take minutes rather than a single pulse time for a pulsed laser, continues wave lasers are able to heat larger areas at once.

Gold nanoshells, coated silica nanoparticles with a thin layer of gold. have been conjugated to antibodies (anti-HER2 or anti-IgG) via PEG linkers. After incubation of SKBr3 cancer cells with the gold nanoshells, an 820 nm laser was used to irradiate the cells. Only the cells incubated with the gold nanoshells conjugated with the specific antibody (anti-HER2) were damaged by the laser. Another category of gold nanoshells are gold layer on liposomes, as soft template. In this case, drug can also be encapsulated inside and/or in bilayer and the release can be triggered by laser light.

thermo Nano-Architectures (tNAs)

The failure of clinical translation of nanoparticles-mediated PTT is mainly ascribed to concerns about their persistence in the body. Indeed, the optical response of anisotropic nanomaterials can be tuned in the NIR region by increasing their size to up to 150 nm. On the other hand, body excretion of non-biodegradable noble metals nanomaterials above 10 nm occurs through the hepatobiliary route in a slow and inefficient manner. A common approach to avoid metal persistence is to reduce the nanoparticles size below the threshold for renal clearance, i.e. ultrasmall nanoparticles (USNPs), meanwhile the maximum light-to-heat transduction is for < 5 nm nanoparticles. On the other hand, the surface plasmon of excretable gold USNPs is in the UV/visible region (far from the first biological windows), severely limiting their potential application in PTT.

Excretion of metals has been combined with NIR-triggered PTT by employing ultrasmall-in-nano architectures composed by metal USNPs embedded in biodegradable silica nanocapsules. t NAs are the first reported NIR-absorbing plasmonic ultrasmall-in-nano platforms that jointly combine: i) photothermal conversion efficacy suitable for hyperthermia, ii) multiple photothermal sequences and iii) renal excretion of the building blocks after the therapeutic action. Nowadays, tNAs therapeutic effect has been assessed on valuable 3D models of human pancreatic adenocarcinoma.

Graphene is viable for photothermal therapy. An 808 nm laser at a power density of 2 W/cm ² was used to irradiate the tumor sites on mice for 5 minutes. As noted by the authors, the power densities of lasers used to heat gold nanorods range from 2 to 4 W/cm ². Thus, these nanoscale graphene sheets require a laser power on the lower end of the range used with gold nanoparticles to photothermally ablate tumors.

In 2012, Yang et al. incorporated the promising results regarding nanoscale reduced graphene oxide reported by Robinson et al. into another in vivo mice study.< The therapeutic treatment used in this study involved the use of nanoscale reduced graphene oxide sheets, nearly identical to the ones used by Robinson et al. (but without any active targeting sequences attached). Nanoscale reduced graphene oxide sheets were successfully irradiated in order to completely destroy the targeted tumors. Most notably, the required power density of the 808 nm laser was reduced to 0.15 W/cm ², an order of magnitude lower than previously required power densities. This study demonstrates the higher efficacy of nanoscale reduced graphene oxide sheets as compared to both nanoscale graphene sheets and gold nanorods.

PTT utilizes photothermal transduction agents (PTAs) which can transform light energy to heat through photothermal effect to raise the temperature of tumor area and thus cause the ablation of tumor cells. Specifically, ideal PTAs should have high photothermal conversion efficiency (PCE), excellent optical stability and biocompatibility, and strong light adsorption in the near-infrared (NIR) region (650-1350 nm) due to the deep-tissue penetration and minimal absorption of NIR light in the biological tissues. PTAs mainly include inorganic materials and organic materials. Inorganic PTAs, such as noble metal materials, carbon-based nanomaterials, and other 2D materials, have high PCE and excellent photostability, but they are not biodegradable and thus have potential long-term toxicity in vivo. Organic PTAs including small molecule dyes and conjugated polymers (CPs) have good biocompatibility and biodegradability, but poor photostability. Among them, small molecule dyes, such as cyanine, porphyrin, phthalocyanine, are limited in the field of cancer treatment because of their susceptibility to photobleaching and poor tumor enrichment ability. Conjugated polymers with large π−π conjugated skeleton and a high electron delocalization structure show potential for PTT due to their strong NIR absorption, excellent photostability, low cytotoxicity, outstanding PCE, good dispersibility in aqueous medium, increased accumulation at tumor site, and long blood circulation time. Moreover, conjugated polymers can be easily combined with other imaging agents and drugs to construct multifunctional nanomaterials for selective and synergistic cancer therapy.

The CPs used for tumor PTT mainly include polyaniline (PANI), polypyrrole (PPy), polythiophene (PTh), polydopamine (PDA), donor−acceptor (D-A) conjugated polymers, and poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) ( PEDOT:PSS).

Photothermal conversion mechanism

The nonradiative process for heat generation of organic PTAs is different from that of inorganic PTAs such as metals and semiconductors which is related with surface plasmon resonance. As shown in the figure, conjugated polymers are first activated to the excited state (S1) under light irradiation and then excited state (S1) decays back to the ground state (S0) via three processes: (I) emitting a photon ( fluorescence), (II) intersystem crossing, and (III) nonradiative relaxation (heat generation). Because these three pathways of the S1 decaying back to the S0 are usually competitive in photosensitive materials, light emitting and intersystem crossing must be efficiently reduced in order to increase the heat generation and improve the photothermal conversion efficiency. For conjugated polymers, on the one hand, their unique structures lead to closed stacking of the molecular sensitizers with highly frequent intermolecular collisions which can efficiently quench the fluorescence and intersystem crossing, and thus enhance the yield of nonradiative relaxation. On the other hand, compared with monomeric phototherapeutic molecules, conjugated polymers possess higher stability in vivo against disassembly and photobleaching, longer blood circulation time, and more accumulation at tumor site due to the enhanced permeability and retention (EPR) effect. Therefore, conjugated polymers have high photothermal conversion efficiency and a large amount of heat generation. One of the most widely used equations to calculate photothermal conversion efficiency (η) of organic PTAs is as follows:

where h is the heat transfer coefficient, A is the container surface area, ΔΤ max means the maximum temperature change in the solution, A λ means the light absorbance, I is the laser power density, and Qs is the heat associated with the light absorbance of the solvent.

Furthermore, various efficient methods, especially donor-acceptor (D-A) strategy, have been designed to enhance the photothermal conversion efficiency and heat generation of conjugated polymers. The D-A assembly system in the conjugated polymers contributes to strong intermolecular electron transfer from the donor to the acceptor, thus bringing efficient fluorescence and intersystem crossing quenching, and improved heat generation. In addition, the HOMO-LUMO gap of the D−A conjugated polymers can be easily tuned through changing the selection of electron donor (ED) and electron acceptor (EA) moieties, and thus D−A structured polymers with extremely low band gap can be developed to improve the NIR absorption and photothermal conversion efficiency of CPs.

Polypyrrole (PPy) is suited for PTT applications because of its strong NIR absorbance, large PCE, stability, and biocompatibility. In vivo experiments show that tumors treated with PPy NPs could be effectively eliminated under the irradiation of an 808 nm laser (1 W cm −2, 5 min). PPy nanosheets exhibit promising photothermal ablation ability toward cancer cells in the NIR II window for deep-tissue PTT.

PPy nanoparticles and its derivative nanomaterials can also be combined with imaging contrast agents and diverse drugs to construct multifunctional theranostic applications in imaging-guided PTT and synergistic treatment, including fluorescent imaging, magnetic resonance imaging (MRI), photoacoustic imaging (PA), computed tomography (CT), photodynamic therapy (PDT), chemotherapy, etc. For example, PPy has been used to encapsulate ultrasmall iron oxide nanoparticles (IONPs) and finally develop IONP@PPy NPs for in vivo MR and PA imaging-guided PTT. Polypyrrole (I-PPy) nanocomposites have been investigated for CT imaging-guided tumor PTT.

Polythiophene (PTh) and its derivatives-based polymers are also one kind of conjugated polymers for PTT. Polythiophene-based polymers usually exhibit excellent photostability, large light-harvesting ability, easy synthesis, and facile functionalization with different substituents.

Conjugated copolymer (C3) with promising photothermal properties can be prepared by linking 2-N,N′-bis(2-(ethyl)hexyl)-perylene-3,4,9,10-tetra-carboxylic acid bis-imide to a thienylvinylene oligomer. C3 was coprecipitated with PEG-PCL and indocyanine green (ICG) to obtain PEG-PCL-C3-ICG nanoparticles for fluorescence-guided photothermal/photodynamic therapy against oral squamous cell carcinoma (OSCC). A biodegradable PLGA-PEGylated DPPV (poly{2,2′-[(2,5-bis(2-hexyldecyl)-3,6-dioxo-2,3,5,6-tetrahydropyrrolo[3,4-c]-pyrrole-1,4-diyl)-dithiophene]-5,5′-diyl-alt-vinylene) conjugated polymer for PA-guided PTT with PCE 71% (@ 808 nm, 0.3 W cm−2). The vinylene bonds in the main chain improves the biodegradability, biocompatibility and photothermal conversion efficiency of CPs.

Dopamine is one of neurotransmitters in the body which helps cells send impulses. Polydopamine (PDA) is obtained through the self-aggregation of dopamine to form a melanin-like substance under mild alkaline conditions. PDA has strong NIR absorption, good photothermal stability, excellent biocompatibility and biodegradability, and high photothermal conversion efficiency. Furthermore, with π conjugated structure and different active groups, PDA can be easily combined with various materials to achieve multifunction, such as fluorescence imaging, MRI, CT, PA, targeted therapy etc. In view of this, PDA and its composite nanomaterials have a broad application prospect in the biomedical field.

Dopamine-melanin colloidal nanospheres is an efficient near-infrared photothermal therapeutic agent for in vivo cancer therapy. PDA can also be modified on the surface of other PTAs, such as gold nanorods, carbon-based materials, to enhance the photothermal stability and efficiency in vivo. For example, PDA-modified spiky gold nanoparticles (SGNP@PDAs) have been investigated for chemo-photothermal therapy.

Donor−acceptor (D−A) conjugated polymers have been investigated for the medicinal purposes. Nano-PCPDTBT CPs have two moieties: 2-ethylhexyl cyclopentadithiophene and 2,1,3-benzothiadiazole. When the PCPDTBT nanoparticle solution (0.115 mg/mL) was exposed to an 808 nm NIR laser (0.6 W/cm ²), the temperature could be increased by more than 30 °C. Wang et al. designed four NIR-absorbing D-A structured conjugated polymer dots (Pdots) containing diketopyrrolo-pyrrole (DPP) and thiophene units as effective photothermal materials with the PCE up to 65% for in vivo cancer therapy. Zhang et al. constructed PBIBDF-BT D-A CPs by using isoindigo derivative (BIBDF) and bithiophene (BT) as EA and ED respectively. PBIBDF-BT was further modified with poly(ethylene glycol)-block-poly(hexyl ethylene phosphate) (mPEG-b-PHEP) to obtain PBIBDF-BT@NP PPE with PCE of 46.7% and high stability in physiological environment. Yang's group designed PBTPBF-BT CPs, in which the bis(5-oxothieno[3,2-b]pyrrole-6-ylidene)-benzodifurandione (BTPBF) and the 3,3′-didodecyl-2,2′-bithiophene (BT) units acting as EA and ED respectively. The D-A CPs have a maximum absorption peak at 1107 nm and a relative high photothermal conversion efficiency (66.4%). Pu et al. synthesized PC70BM-PCPDTBT D-A CPs via nanoprecipitation of EA (6,6)-phenyl-C71-butyric acid methyl ester (PC70BM) and ED PCPDTBT (SPs) for PA-guided PTT. Wang et al. developed D-A CPs TBDOPV-DT containing thiophene-fused benzodifurandione-based oligo(p-phenylenevinylene) (TBDOPV) as EA unit and 2,2′-bithio-phene (DT) as ED unit. TBDOPV-DT CPs have a strong absorption at 1093 nm and achieve highly efficient NIR-II photothermal conversion.

Poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) (PEDOT:PSS) is often used in organic electrons and have strong NIR absorption. In 2012, Liu's group first reported PEGylated PEDOT:PSS polymeric nanoparticle (PEDOT:PSS-PEG) for near-infrared photothermal therapy of cancer. PEDOT:PSS-PEG nanoparticles have high stability in vivo and long blood circulation half-life of 21.4 ± 3.1 h. The PTT in animals showed no appreciable side effects for the tested dose and an excellent therapeutic efficacy under the 808 nm laser irradiation. Kang et al. synthesized magneto-conjugated polymer core−shell MNP@PEDOT:PSS nanoparticles for multimodal imaging-guided PTT. Furthermore, PEDOT:PSS NPs can not only serve as PTAs but also as a drug carrier to load various types of drugs, such as SN38, chemotherapy drugs DOX and photodynamic agent chlorin e6 (Ce6), thus achieving synergistic cancer therapy.

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Most materials of interest currently being investigated for photothermal therapy are on the nanoscale. One of the key reasons behind this is the enhanced permeability and retention effect observed with particles in a certain size range (typically 20 - 300 nm). Molecules in this range have been observed to preferentially accumulate in tumor tissue.

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Children's Health Defense® is a 501(c)(3) non-profit organization. Its mission is to end childhood health epidemics by working aggressively to eliminate harmful exposures, hold those responsible accountable, and to establish safeguards to prevent future harm. Donations are tax deductible to the full extent of the law.

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Blood, breathing...Is creatine kinase associated with outcome in COVID-19?

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Views: 12717 | Downloads: 375 | Cited: 5

Views:12717 | Downloads:375 | Cited: 5 |

Academic Editor: Athanassios P. Kyritsis | Copy Editor: Cai-Hong Wang | Production Editor: Jing Yu

The new "severe acute respiratory syndrome" (SARS)-correlated coronavirus (SARS-CoV-2), first isolated from three patients from Wuhan (China), has been spreading worldwide since the end of 2019, emerging as an exceptional global crisis. This disorder was called "coronavirus disease 2019" or COVID-19. Clinical features are highly variable, and the clinical severity ranges from asymptomatic to fatal. The understanding of SARS-CoV-2 infection is ongoing. Several neurological symptoms were identified as part of the COVID-19 spectrum, including muscle pain, confusion, and headache.

A study performed on 113 patients who deceased from COVID-19 revealed that acute respiratory distress syndrome with respiratory failure, sepsis, heart failure and subsequently hypoxic encephalopathy were the most common severe complications. The median age of the deceased patients was 68 years and male gender was predominant (73%). Hypertension and other cardiovascular disorders were more frequent among the patients who died. Dyspnea, chest tightness and altered consciousness were more frequent in the deceased patients, as well as hyperCKemia (increased levels of creatine kinase, CK).

Other retrospective studies provided partly similar results regarding CK, but data on this marker of muscle damage were only briefly mentioned in most papers, and a detailed literature review is not available to date. This exploratory literature review aimed to summarize early findings on the relevance of CK as a marker potentially associated with outcome in patients infected by Sars-CoV-2.

PubMed was searched up to May 13, 2020 for all articles about "(sars* OR covid* OR coronavirus) and (CK OR CPK OR creatine kinase OR creatine phosphokinase OR muscular OR muscle OR retrospective OR series)", and the abstracts were reviewed to identify all the relevant publications. The full text of all the retrospective case series or cohort studies which were identified according to this search were then screened for the following words: "kinase" or "CK" or "CPK". All the studies reporting data on CK levels (in any form) were included in this analysis and summarized in Table 1. Retrospective studies including only children or pregnant women were not considered. We mainly focused on the publications where there was a distinction between less severe and more severe cases (defined in any possible way, see Table 1) which could work as an outcome measure. These references, allowing an evaluation of CK as a marker potentially associated with outcome, are underscored in Table 1.

Author

Country

Definition of severe cases

Severe/total cases

Mean age (or median)

Males (%)

How CK is reported

When blood was sampled

Results (concerning CK)

China

ICU/mechanical ventilation or death
("composite end-point")

32/95

49 (median)

56%

% of cases with CK < 200 (normal) or 200-400 or 400-600 or >600 U/L

Highest levels during hospitaliza-tion

For < 200 U/L group, 17.9% of the patients reached the composite endpoint. For 200-400 U/L group, 41.7% of the patients reached the composite endpoint. For 400-600 U/L group, 44.4% of the patients reached the composite endpoint. For > 600 U/L group, 57.1% of the patients reached the composite endpoint. (sign.)
Higher CK activity correlated strongly with severe pneumonia and composite endpoint. (sign.)

China

Radiological evidence of pneumonia

573/ 665

47 (pneumonia)
35 (others)
(sign.)

51%

Continous value (U/L)

?

Mean CK 73 U/L (patients with pneumonia) vs. 63 U/L (others) (sign.)
In the pneumonia group, higher CK could predict a more severe clinical picture (univariate OR 1.001 with confidence interval 1.000-1.002) (sign.)

China

More severe respiratory insufficiency

30/161

45 (median)

50%

Both continuous values (U/L) and % of cases with CK > 190 U/L

Admission

Median CK higher in severe patients (100 vs. 69 U/L) (sign.)
HyperCKemia present in 30% of the more severe cases vs. 6% of the others (sign.)

China

Death

54/191

56 (median)

62%

Both continuous values (U/L) and % of cases with CK > 185 U/L

Admission

Median CK overall 21.5 U/L: 39 U/L in the patients who died and 18 U/L in subjects who were discharged (sign.)
HyperCKemia (> 185 U/L): 22/168 (13%) overall: 11/52 (21%) in deceased subjects vs. 11/116 (9%) of the other patients (sign.). HyperCKemia was associated with death: univariate OR (95%CI) 2.56 (1.03-6.36) (sign.)

Twenty-one retrospective studies which included data on CK were identified [Table 1]. Among these 21 papers, 12 (underscored in Table 1) provided a measure of severity potentially useful to serve as an outcome measure. Eleven of these studies were from China and one from South Korea.

There is marked heterogeneity between these studies. In some instances, CK data were provided as a continuous variable, whereas other studies showed the fraction of patients with CK higher than a given value (different from study to study). In some cases, CK levels were presented as a mean, in others as a median. Furthermore, the definition of severe cases, and their percentage, mean (or median) age and gender proportion were also markedly different from study to study. Blood was sampled at admission in most of these retrospective studies (but not in all of them). These evident differences precluded further statistical analysis and a formal meta-analysis.

However, some conclusions could be drawn. In 11 of the 12 studies considered, CK levels were higher in the more severe group of COVID-19 patients. Among these 11 studies, the difference reached statistical significance in 7 studies, while there was no statistical significance in 4. The significant studies included an average of 249 patients, compared with 69 patients in the other studies. Therefore, the lower power of the smaller studies could explain the fact that the difference did not reach statistical significance in some of these studies.

The five larger studies, each involving more than 160 patients, showed similar findings.

Chen and coworkers observed that CK levels at admission were markedly higher in the patients who subsequently died (median 189 U/L) compared with the ones who recovered (median 84 U/L).

Mao and coworkers reported that CK was higher in the patients with more severe pneumonia; unfortunately, this study did not explicitly specify when CK was tested.

Zhang et al. reported that mean CK was higher in the patients with pneumonia compared with the patients with no radiological evidence of pneumonia; in the pneumonia group, higher CK could predict a more severe clinical picture.

Zheng et al. showed that median CK levels at admission were higher in the more severe group; hyperCKemia (> 190 U/L) was present in 30% of the more severe cases vs. 6% of the others (significant difference).

Zhou et al. observed that median CK at admission was higher in the patients who subsequently died than in the subjects who were discharged; hyperCKemia (> 185 U/L) was observed in 21% of the deceased persons compared with 9% of the others, and was associated with death (univariate odds ratio 2.56, 95% confidence interval 1.03-6.36).

Taken together, the retrospective studies available to date (especially the larger ones) showed some association between CK levels and the clinical outcome of patients infected by SARS-CoV-2. CK was determined at hospital admission in most cases.

This preliminary review showed the limitations typical of all the systematic reviews based on retrospective studies. It is not possible to rule out the influence of various confounding factors in the original studies. Some of the included studies did not fully clarify inclusion criteria, course of disease and severity of disease. Lastly, most of these studies were performed in China, and this could lead to geographic and ethnicity biases.

Furthermore, data on CK levels were only briefly mentioned in most papers, and possible correlations with other clinical features (such as muscle pain) were not researched.

Only the retrospective study performed in Wuhan reported that "skeletal muscle injury" (defined in the following way: "when a patient had skeletal muscle pain and elevated serum CK", greater than 200 U/L) was significantly more frequent in severe COVID-19, compared with less severe diseases (19% vs. 5%). Median CK levels were higher in the more severe group: 83 U/L (range 9-12,216) vs. 59 U/L (19-1,260). Interestingly, patients with "muscle injury" had multiorgan damage, including more severe liver and kidney abnormalities.

Another Chinese retrospective work performed on compromised patients reported that muscle pain was an onset symptom in 22%. Interestingly, a European study performed on patients with much milder symptoms reported a higher prevalence of muscle pain (63%).

Furthermore, severe rhabdomyolysis is a possible, rare complication of COVID-19.

A third of patients with other coronavirus infections (namely SARS) have manifested myalgia and elevated CK, and in some instances rhabdomyolysis. Therefore, even if electromyography (EMG), muscle imaging or muscle histopathology is not available to date, coronavirus infections may possibly cause viral myositis on the basis of the above reported data. Immune-mediated mechanisms should also be considered. Furthermore, severely ill patients may develop weakness due to muscle atrophy from disuse and/or critical illness myopathy (and/or polyneuropathy), so specifically designed studies are needed.

The most common neurological features associated with COVID-19 are presented in Table 2. Muscle pain is common in both mild and severe cases, and in the most compromised patients, it is accompanied by increased CK levels and possibly true myopathic damage.

Neurological features typical of mild/initial cases

Neurological features typical of severe cases

Headache
Smell impairment
Myalgia/muscle pain

Delirium
Impaired consciousness
Pyramidal signs
Ischemic stroke (rare)
Muscle damage with increased CK

Interestingly, angiotensin converting enzyme 2, which was identified as the functional receptor for SARS-CoV-2, is present in multiple human tissues, including skeletal muscle and nervous system. Therefore, the longitudinal follow-up of people who have been infected by SARS-CoV-2 should include a careful assessment of the neuromuscular system. There is a strong need for prospective and specifically designed studies, including EMG, muscle imaging, muscle histopathology and PCR assay for detection of SARS-CoV-2 in muscle tissue.

In conclusion, some association between CK levels and the clinical outcome of patients infected by SARS-Cov-2 seems to exist, but the precise mechanisms are still unknown. The influence of this novel coronavirus on voluntary muscle really needs to be clarified.

Declarations

Authors' contributions

The author contributed solely to the article.

Availability of data and materials

Not applicable.

Financial support and sponsorship

None.

Conflicts of interest

The author declared that there are no conflicts of interest.

Ethical approval and consent to participate

Not applicable.

Consent for publication

This Ediotrial can be published in the present form.

© The Author(s) 2021.

References

5. Chen X, Yang Y, Huang M, et al. Differences between COVID-19 and suspected then confirmed SARS-CoV-2-negative pneumonia: a retrospective study from a single center. J Med Virol 2020; doi: 10.1002/jmv.25810.
DOI PubMed

17. Zangrillo A, Beretta L, Scandroglio AM, et al. Characteristics, treatment, outcomes and cause of death of invasively ventilated patients with COVID-19 ARDS in Milan, Italy. Crit Care Resusc 2020.
PubMed

20. Zheng F, Tang W, Li H, et al. Clinical characteristics of 161 cases of corona virus disease 2019 (COVID-19) in Changsha. Eur Rev Med Pharmacol Sci 2020;24:3404-10.
DOI PubMed

24. Guidon AC, Amato AA. COVID-19 and neuromuscular disorders. Neurology 2020;94:959-69.
DOI PubMed

25. Leung TW, Wong KS, Hui AC, et al. Myopathic changes associated with severe acute respiratory syndrome: a postmortem case series. Arch Neurol 2005;62:1113-7.
DOI PubMed

Cite This Article

Orsucci D. Is creatine kinase associated with outcome in COVID-19?. Neuroimmunol Neuroinflammation 2021;8:216-21. http://dx.doi.org/10.20517/2347-8659.2020.53

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Is creatine kinase associated with outcome in COVID-19?

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Views: 12717 | Downloads: 375 | Cited: 5 Views:12717 | Downloads:375 | Cited: 5 | Academic Editor: Athanassios P. Kyritsis | Copy Editor: Cai-Hong Wang | Production Editor: Jing Yu The new "severe acute respiratory syndrome" (SARS)-correlated coronavirus (SARS-CoV-2), first isolated from three patients from Wuhan (China), has been spreading worldwide since the end of 2019, emerging as an exceptional global crisis.

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