IV. Mechanobiology/Mechanotransduction/Mechanotherapy
Mechanobiology:
Cells in our body actively sense and respond to a variety of mechanical signals. The mechanical stiffness of the surrounding extracellular matrix (ECM) critically determines normal cell function, stem cell differentiation and tissue homeostasis. Abnormal changes in ECM stiffness contribute to the onset and progression of various diseases, such as cancer and fibrosis.
Cells often experience forces in the form of shear stress during breathing and blood flow, compression and tension due to muscle contraction. Forces play a crucial role in regulating tissue morphogenesis in developing embryos. The sensitivity of cells to forces and substrate stiffness has been recognized as a powerful tool in tissue engineering, where it can be harnessed to design biomaterials that optimally guide stem cells or resident cells in the patient towards generating a functional replacement tissue. Given its central importance in cell function and human health, mechanobiology has emerged as a new and growing field that attracts researchers from disciplines ranging from cell and developmental biology, to bioengineering, material science and biophysics.
A central element in mechanobiology is cellular “mechanosensing”. Cells actively probe the rigidity of their extracellular environment by exerting traction forces via transmembrane proteins (integrins). It is still poorly understood how probing by traction forces allows cells to sense matrix stiffness and how cells transduce this mechanical information into a cellular response. Most experimental studies until now were performed with cells cultured on top of two dimensional (2D), and often rigid, substrates, which inadequately mimic most physiological contexts.
Mechanosensing and mechanotransduction are cellular processes that involve both intra- and extracellular components.
The main structural components that contribute are:
- integrins
- the extracellular matrix
- the intracellular cytoskeleton.
Mechanical forces and biochemical signaling are integrated by various intracellular signaling pathways.
The fundamental aspects of cell behavior are mechanosensitive, including adhesion, spreading, migration, gene expression and cell-cell interactions. Substrate stiffness can regulate stem cell differentiation and compete with biochemical cues. Recent experiments with stem cells on photodegradable substrates showed, stem cells remember the mechanical history of their environment. It is unclear whether cells sense their environment by applying a constant stress (eg. force) and reading out the strain (eg. deformation) or vice versa. Theoretical models suggest, cells may readjust their contractile activity and cytoskeleton organization to maintain either an optimal strain or an optimal stress.
Mechanobiology within our cells: the 3D fibrous extracellular network composition of integrins, ECM, important signaling pathways and cytoskeleton.
(Jansen et al, 2015)
What happens at the tissue level to promote repair and remodelling of tendon, muscle, articular cartilage and bone? = Mechanotransduction
Mechanotransduction is the physiological process where cells sense and respond to mechanical loads.
Mechanotransduction is generally broken down into three steps:
- mechanocoupling
- cell–cell communication
- the effector response.
To simplify, these elements/steps can be thought of as:
1. the mechanical trigger or catalyst
2. the communication throughout a tissue to distribute the loading message
3. the response at the cellular level to effect the response - that is, the tissue ‘‘factory’’ that produces and assembles the necessary materials in the correct alignment.
Communication at each stage occurs via cellular signalling - an information network of messenger proteins, ion channels and lipids.
**These fundamental processes also apply to other musculoskeletal tissues**
Mechanocoupling refers to physical load (often shear or compression) causing a physical perturbation to cells that make up a tissue. Tensile and shearing forces elicit a deformation of the cell that can trigger a wide array of responses depending on the type, magnitude and duration of loading.
Consider the skeleton as an example of a connective tissue; the body’s sensor is the osteocyte network and the process of regulating bone to load has been referred to as the ‘’mechanostat”. In the absence of activity, the mechanotransduction signal is weak, so connective tissue is lost (eg, osteoporosis). When there are loads above the tissue’s set point, there is a stimulus through mechanotransduction so that the body adapts by increasing protein synthesis and adding tissue where possible (larger, stronger bone).
Mechanotransduction requires a mechanical signal to be transmitted to the microenvironment of a cell and for the cell to possess machinery to sense the signal. Cells can be exposed to a variety of micromechanical stimuli, with the precise nature of the stimulus depending on the mechanical properties of the cells themselves and the interaction between the incoming mechanical signal and the extracellular matrix (ECM). Common stimuli include tension, compression, and shear; however, cells also can be exposed to other mechanical stimuli, such as hydrostatic pressure, vibration, and fluid shear.
The tissue in which a cell resides and the location of the cell within that tissue influence the forces to which the cell is exposed; yet, the exact nature of the forces may not always be evident. For example, it may be assumed that mechanosensitive cells in bone are predominantly exposed to compression, whereas those in tendon are exposed to tension due to the function of the tissues in which they reside. However, long bones (eg, tibia and femur) are curved and bend when axially loaded to generate compressive forces within the tissue on the side the bone is bending toward and tensile stresses within the contralateral side. Bone cells can be exposed to either compressive or tensile forces (although fluid shear appears to be the most likely signal involved in skeletal mechanotransduction [discussed later]). Similarly, although tendons are exposed to large tensile forces in their role of transmitting muscle forces, the tensile loading of collagen can cause cell-occupying spaces to narrow, resulting in the generation of compressive forces, whereas differential elongation of adjacent collagen fibers can generate microscopic shearing forces. Cells located in tendon near bony prominences (eg, within the supraspinatus tendon as it passes through the subacromial space or the Achilles tendon near its calcaneal insertion) can be exposed principally to compressive, rather than tensile, forces.
By understanding the forces to which cells are exposed and respond, it may be possible to develop novel means of introducing those forces to induce a desired cellular response and resultant tissue adaptation. In particular, it may be possible to encourage the commitment of endogenous adult stem and progenitor cells to a particular lineage to enhance regenerative potential. There is a reciprocal relationship between cells and tissue during development wherein the tissue type influences the forces to which cells are exposed, while forces determine cellular differentiation and subsequently what tissue type is produced.18 By introducing specific forces at specific times, resident regenerative cells can be encouraged to commit to a specific lineage and produce a particular tissue type.
It is possible that the ECM-integrin-cytoskeleton axis principally acts to alter the mechanosensitivity of a cell by changing the cell's internal stiffness and how much it pulls on the surrounding ECM, it also is possible that the actual conversion of a mechanical stimulus into a molecular response (ie, mechanotransduction) is primarily mediated by conformational changes in transmembrane mechanosensitive proteins. These proteins include stretch-activated ion channels, cell membrane spanning G-protein-coupled receptors, and growth factor receptors. Any changes in normal intracellular force transmission through changes in intracellular or ECM structure and organization may lead to altered forces acting on these proteins, resulting in a change in their affinity to binding partners or ion conductivity and the initiation of downstream molecular signaling pathways.
Tendons:
Tendon is a dynamic, mechanoresponsive tissue. One of the major load-induced responses shown both in vitro and in vivo in tendon is an upregulation of insulin-like growth factor (IGF-I). This upregulation of IGF-I is associated with cellular proliferation and matrix remodelling within the tendon. However, recent studies suggest that other growth factors and cytokines in addition to IGF-I are also likely to play a role. Alfredson et al (2004) examined tendon structure by grey-scale ultrasound in 26 tendons with Achilles tendinosis, which had been treated with eccentric exercise. Remarkably, after a mean follow up of 3.8 years, 19 of 26 tendons had a more normalized structure, as gauged by their thickness and by the reduction of hypoechoic areas. This study and others show that tendon can respond favourably to controlled loading after injury. Research into the ideal loading conditions for different types of tendon injury is still ongoing.
Muscle:
Muscle is highly responsive to changes in functional demands through the modulation of load- induced pathways. Overload leads to the immediate, local upregulation of mechanogrowth factor (MGF), a splice variant of IGF-I with unique actions. MGF expression in turn leads to muscle hypertrophy via activation of satellite cells. The clinical application of mechanotherapy for muscle injury is based on animal studies. After a brief rest period to allow the scar tissue to stabilize, controlled loading is initiated. The benefits of loading include: improved alignment of regenerating myotubes, faster and more complete regeneration, and minimisation of atrophy of surrounding myotubes.
Articular Cartilage:
Articular cartilage is populated by mechanosensitive cells (chondrocytes), which signal via highly analogous pathways.
**57 consecutive patients with isolated full-thickness cartilage defect of the patella and disabling knee pain of long duration by periosteal transplantation either with or without continuous passive motion (CPM). In this study, 76% of patients using CPM achieved an ‘‘excellent’’ outcome, whereas only 53% achieved this in the absence of CPM. Tissue repair was not directly assessed in this case series, but the results encourage further research into the underlying tissue response and the optimization of loading parameters. **
To effectively stimulate healing, combine light traction to decompress the affected joint(s) and facilate collagen remodelling.
Collagen:
Collagen is a protein which is a major constituent of the extracellular matrix of connective tissue. It is the main load carrying element in a wide variety of soft tissues and is very important to human physiology (for example, the collagen content of (human) achilles tendon is about 20 times that of elastin). Collagen is a macromolecule with length of about 280 nm. Collagen molecules are linked to each other by covalent bonds building collagen fibrils. Depending on the primary function and the requirement of strength of the tissue the diameter of collagen fibrils varies (the order of magnitude is 1.5 nm). In the structure of tendons and ligaments, for example, collagen appears as parallel oriented fibers, while many other tissues have an intricate disordered network of collagen fibres embedded in a gelatinous matrix of proteoglycans (Nimni et al, 1988; Betsch et al, 1980).
More than 12 types of collagen have been identified (Nimni et al, 1988). The most common collagen is type I, which can be isolated from any tissue. It is the major constituent in blood vessels. The rod-like shape of the collagen molecule comes from three polypeptide chains which are composed in a right-handed triple-helical conformation. Most of the collagen molecule consists of three amino acids; glycine (33%), which enhances the stability of the molecule, proline (15%) and hydroxyproline (15%) (Ramachandran, 1967).
The intramolecular crosslinks of collagen gives the connective tissues the strength which varies with age, pathology, etc. (for a correlation between the collagen content in the tissue, % dry weight, and its ultimate tensile strength. The function and integrity of organs are maintained by the tension in collagen fibers. They shrink upon heating due to breakdown of the crystalline structure (at 65 C, mammalian collagen shrinks to about one-third of its initial length).
Elastin:
Elastin, like collagen, is a protein which is a major constituent of the extracellular matrix of connective tissue. It is present as thin strands in soft tissues such as skin, lung, ligamentum flava of the spine and ligamentum nuchae (the elastin content of the latter is about 5 times that of collagen).
The long flexible elastin molecules build up a three-dimensional (rubber-like) network, which may be stretched to about 2.5 of the initial length of the unloaded configuration. In contrast to collagen fibers, this network does not exhibit a pronounced hierarchical organization. As for collagen, 33% of the total amino acids of elastin consists of glycine. However, the proline and hydroxyproline contents are much lower than in collagen molecules.
The mechanical behavior of elastin may be explained within the concept of entropic elasticity.
As for rubber, the random molecular conformations, and hence the entropy, change with deformation.
Elasticity arises through entropic straightening of the chains, i.e. a decrease of entropy, or an increase of internal energy. Elastin is essentially a linearly elastic material; small relaxation effects (Hoeve, 1958).
Regarding the modeling of strain recovery that we measured, simple deterministic models of collagen intermolecular interactions that take the fibrils beyond their yield point would not be expected to display strain recovery upon unloading. However, molecular dynamics simulations that contain temperature effects may well show this behavior. Buehler has not published unloading or cyclic loading curves using his multiscale model that uses a molecular dynamics simulation to model collagen molecule conformations. It would be very interesting to see if these temperature dependent effects result in strain recovery upon unloading. Another possible physical source for the strain hardening and strain recovery we measured may be related to the movement of water in the system. If loading the sample in tension results in a reduction in fibril radius, this could create an internal pressure that would drive water out of the fibril, making it stiffer. Unloading the fibril might allow the water back into the fibril to some extent, causing a recovery of the original state. (Shen et al 2008)
Bone:
In bone, osteocytes are the primary mechanosensors. A recent clinical study suggested that the beneficial effect of mechanotransduction may be exploited by appropriately trained physical therapists to improve fracture healing. In this study, 21 patients with a distal radius fracture were randomized to receive standard care including immobilisation and gripping exercises or standard care plus intermittent compression delivered via an inflatable pneumatic cuff worn under the cast. The experimental group displayed significantly increased strength (12–26%) and range of motion (8–14%) at the end of the immobilisation period and these differences were maintained at 10 weeks. Future, larger studies are planned by this group to confirm whether the effects of compression affected the fracture healing itself, as suggested by preclinical studies with similar loading parameters.
Regenerative medicine has opened the possibility for full healing of injured or degenerated musculoskeletal tissues, thereby offering hope for people who have conditions that traditionally have had limited recovery potential. Examples of musculoskeletal conditions that may benefit from regenerative medicine approaches include:
- injury-related conditions that use repair processes to heal, such as muscle strains, ligament sprains, tendon ruptures, and integument wounds
- injury-related conditions that exhibit compromised healing, such as osteochondral defects and nonunited bone fractures
- injury-related conditions that have little prospect of healing, such as volumetric muscle loss and segmental bone defects
- disease-related conditions, such as sarcopenia, osteoporosis, and osteoarthrosis.
Currently the use of stem cells, progenitor cells, or biologically active molecules and the implantation of bioengineered scaffolds or ex vivo grown tissues, are being researched and in some cases applied into clinical practice.
As the goal of regenerative medicine is to restore or establish normal function, individuals who receive regenerative therapies will require rehabilitation to make best use of their restored anatomy and newly regained abilities. Physical therapists are specifically trained to assess and manage musculoskeletal pathologies and thus are well positioned to be important allies in musculoskeletal regenerative medicine. However, the role of physical therapists extends beyond the serial approach of simply re-establishing full function following tissue healing.
Musculoskeletal Regenerative Rehabilitation:
Musculoskeletal regenerative rehabilitation can be defined as the integration of principles and approaches from rehabilitation and regenerative medicine, with the ultimate goal of promoting the restoration of function through musculoskeletal tissue regeneration and repair.4 This definition does not confine the role of physical therapists to restoring function after tissue regeneration or repair but also enables therapists to play an active role by facilitating regeneration and repair at the tissue level during healing. In addition, the definition encourages therapists to contribute to the conception and development of novel regenerative therapies by working collaboratively with other disciplines involved in regenerative medicine in a team-based approach to optimize functional outcomes.
The success of therapies in regenerative medicine at repairing or regenerating musculoskeletal tissues ultimately depends on the therapies being accepted and incorporated into the native tissue (eg, in the case of ex vivo grown tissues or bioengineered scaffolds) and creating a musculoskeletal tissue with optimized mechanical characteristics (eg, in the case of biologic or pharmaceutical agents). One group of therapies that physical therapists have in their repertoire that have great potential of having additive, or even synergistic, effects when introduced in conjunction with regenerative medicine treatments is mechanotherapies.
Physical Therapy Example: Regenerative Rehab Perspective
An example of where the microscopic force that cells are exposed to has been partly deconstructed to develop potential novel mechanotherapies is in bone. There is general consensus that osteocytes embedded throughout the bone matrix are the mechanosensors within the skeletal system. Physical deformation (strain) of the bone matrix is not sufficient to deform the osteocyte cell membrane and initiate a response; however, axial compression and bending increase intramedullary pressure, inducing the flow of interstitial fluid from areas of high pressure (compression) to low pressure (tension) within the lacunocanalicular network housing osteocytes and their dendritic processes. Although extravascular pressure drives a baseline flow of interstitial fluid, flow is heightened by the superimposition of intermittent mechanical loading and exposes osteocytes to fluid flow shear forces. A small level of tissue strain induces enhanced shear at the cell membrane, enhancing the mechanical stimulus engendered to the cells.
Based on the purported mechanical milieu that osteocytes are exposed to during tissue loading (ie, fluid flow shear forces) and the observation that pressurization of the intramedullary cavity causes an outward pressure gradient that induces interstitial fluid flow, investigators have begun exploring how to exogenously enhance intramedullary pressure in the absence of significant tissue loading. Example interventions currently in preclinical development include oscillatory muscle stimulation, dynamic flow stimulation, and dynamic joint loading. Each of these modalities increases intramedullary pressure in preclinical animal models to induce bone adaptation, with very negligible tissue loading. Importantly, as the intramedullary cavity contains both hematopoietic cells and MSCs, which are responsive to hydrostatic pressure and fluid flow shear forces, induction of altered intramedullary pressure and interstitial fluid flow via exogenous means has the potential to contribute to regenerative processes. Ultimately, the intramedullary pressure and interstitial fluid flow modalities need to be scaled up to humans before their clinical utility can be realized, but they provide an example of how the microscopic force to which cells are exposed and respond can be developed into potential novel mechanotherapies.
Osteocytes are uniquely positioned to sense mechanical forces; however, MSCs in the bone marrow also perceive force, particularly direct membrane strain, as opposed to the fluid shear stress experienced predominantly by osteocytes.46 As bone marrow MSCs can differentiate into a variety of tissue types, including fat, cartilage, tendon, and bone, understanding the optimal loading parameters to direct lineage commitment is critical for the incorporation of physical stimuli into engineered tissue components. An ex vivo study has shown that direct membrane strain restricts MSC adipogenesis, providing a larger pool of precursor cells available for differentiation toward cartilage, bone, or tendon. These concepts have been carried over to the development of engineered cartilage grafts, where dynamic compression may enhance formation and mechanical competence of cartilaginous grafts.
Low-intensity vibration (LIV) also has been developed as a means of mechanically stimulating musculoskeletal cells in the absence of appreciable tissue deformation forces. Low-intensity vibration evolved from the observation that skeletal muscle not only imparts force on bone during locomotion to engender high strain magnitudes but also generates low-magnitude (<100 microstrain [με]), high-frequency (30- to 90-Hz) stimuli. It induces neither strain49 nor fluid shear50 and thus requires a distinct mechanism for the vibration signals to mediate cellular effects. It has been proposed that LIV induces acceleration of the cell nucleus, which may activate mechanosensitive signaling pathways, as the nucleus is tethered by the internal actin cytoskeleton. In support of this proposed mechanism, it was recently shown that LIV-induced activation of mechanosensitive pathways is reduced by physically disconnecting the nucleus from the supporting actin cytoskeleton.
Unfavourable Reactions: NSAIDs
Physical therapists should explore not only the beneficial interactions induced by combining mechanotherapies and biologically active compounds but also unfavorable interactions. One such interaction is a pathway activated in a range of musculoskeletal cells involving the rapid increase in intracellular calcium concentration, induction of cyclo-oxygenase-2 expression, and release of prostaglandin E2. Interference of this early signaling cascade by introduction of calcium channel blockers, nonsteroidal anti-inflammatory drugs (NSAIDs), or other compounds prior to the delivery of a mechanotherapy may negatively affect adaptive responses. For instance, animal studies have demonstrated that bone formation is substantially blunted when NSAIDs are administered prior to the introduction of a mechanical stimulus. Similarly, a recent randomized controlled trial suggested that individuals taking NSAIDs prior to exercise exhibited an impaired skeletal adaptive response. These bone-related findings are supported by studies in muscle and tendon showing that NSAID administration prior to exercise may reduce adaptive responses.
Mechanotherapy:
Musculoskeletal tissues are critical for load bearing but also generate, absorb, and transmit force, thereby enabling functional movement. Given their mechanical role, it follows teleologically that musculoskeletal tissues are capable of responding and adapting to their mechanical environment. Mechanical forces direct cellular activities influencing the tissue-level processes of growth, modeling, remodeling, and repair, with the ultimate outcomes being altered tissue mass, structure, and quality. Nearly every physical therapy intervention in musculoskeletal rehabilitation introduces mechanical forces, regardless of whether the forces are generated extrinsically via therapist intervention (eg, during joint or tissue mobilization or via the introduction of external therapeutic modalities) or intrinsically within the individual themselves via the prescription of exercise therapy. As an exhaustive review of the numerous forms of mechanotherapies used by physical therapists is outside the scope of the current perspective article, readers are referred to the following reviews that detail various forms of mechanical interventions, including: joint mobilizations, muscle or tendon stretching,6 resistance exercises, vibration platforms, laser therapy aka (PBMT) and massage.
Historical perspective:
The first formal definition of mechanotherapy was published in 1890 as “the employment of mechanical means for the cure of disease.” The term remained relatively unchanged until it was updated in 2009 to “the employment of mechanotransduction for the stimulation of tissue repair and remodeling.” The revised description highlighted the cellular basis of tissue responses and the distinction between healthy and injured tissues. More recently, the definition was again updated to reflect the influence of mechanotherapy on tissues outside of the musculoskeletal system; “any intervention that introduces mechanical forces with the goal of altering molecular pathways and inducing a cellular response that enhances tissue growth, modeling, remodeling, or repair.” As such, we seek to highlight the multisystem hierarchy (molecules – cells – tissues), which is responsive to mechanical signals, and to recognize the influence of mechanotherapy on the tissue-level processes responsible for the development, maintenance, healing, and regeneration of tissues. Additionally, although musculoskeletal tissues are the primary focus, it is important to acknowledge that essentially every cell type within the body is responsive to mechanical signals, extending the principles of mechanotherapy to nonmusculoskeletal tissues.