The cellular mechanobiology of aging: from biology to mechanics

Abstract

Aging is a chronic, complicated process that leads to degenerative physical and biological changes in living organisms. Aging is associated with permanent, gradual physiological cellular decay that affects all aspects of cellular mechanobiological features, including cellular cytoskeleton structures, mechanosensitive signaling pathways, and forces in the cell, as well as the cell’s ability to sense and adapt to extracellular biomechanical signals in the tissue environment through mechanotransduction. These mechanobiological changes in cells are directly or indirectly responsible for dysfunctions and diseases in various organ systems, including the cardiovascular, musculoskeletal, skin, and immune systems. This review critically examines the role of aging in the progressive decline of the mechanobiology occurring in cells, and establish mechanistic frameworks to understand the mechanobiological impacts of aging on disease progression and develop new strategies to halt and reverse the aging process. This review also highlights the recent development of novel bioengineering approaches for studying the key mechanobiological mechanisms in aging.

Introduction

Aging is a chronic, complicated process that leads to functional deterioration in the human body. It is accompanied by accumulated biological and biomechanical changes across all length scales that affect not only the vitality and functional performance of different tissues and organs but also individual cells and subcellular organelles. Particularly, aging represents a permanent, gradual physiological cellular decay, both with biological degradation, such as DNA damage, decreased cellular replication, and accumulation of metabolic damage,¹ and with deterioration in the mechanical properties of the cell² and the accompanying extracellular matrix (ECM).³ In addition to the normal intrinsic aging processes, pathological aging associated with poor lifestyle choices and with diseases such as the Alzheimer’s dementia, cancer, diabetes, and cardiovascular disease cause more severe changes in the human body.⁴ These changes during the normal and pathological aging processes affect various aspects of cellular mechanobiology, including cell mechanics, subcellular cytoskeleton structures, nuclear mechanics, mechanosensitive signaling, and forces in the cell, as well as the cell’s ability to adapt to extracellular mechanical signals in the tissue environment (Fig. 1).⁵ To date, it is still not known exactly what causes these observed mechanobiological changes during the normal and pathological aging processes, but an understanding of the underlying mechanisms is now beginning to emerge.

Figure 1.

Aging-associated alterations in cell mechanobiology, including the ECM, cell mechanics, cytoskeleton, nuclear mechanics, forces, and mechanosensitive signaling.

The ability of the cell to sense the mechanical inputs in the environment and translate them into biochemical signals is called “mechanotransduction” and is the basis for a plethora of cellular activities that support the basic functions of human tissue and organ systems.⁶ Thus, it is paramount to scrutinize how the long-term aging process dysregulates the critical transient mechanosensing and mechanotransductive mechanisms at subcellular and molecular scales, resulting in aging-associated diseases. Moreover, mechanical forces play important roles in the regulation of various biological processes at the molecular and cellular level, such as gene expression, adhesion, migration, and cell fate, which are essential to the maintenance of tissue homeostasis.⁷ The changes in cell cytoskeleton structures, mechanosensitive signaling, and the ECM environment during the aging process dysregulate the kinetics of the intracellular and intercellular forces in various types of tissue cells, which results in aging-related dysregulation and diseases.⁸ These changing mechanobiological characteristics thus might serve as new biomarkers to evaluate the aging process and treat pathological aging-related diseases.

Aging-associated mechanobiological dysregulation largely affects the functions of the cardiovascular,⁹ musculoskeletal,¹⁰ skin,¹¹ and immune systems,¹² and are reflected in the increase of the risk of many diseases. For instance, aging has been linked with an increase in the risk of cardiovascular diseases, where the latest data suggests that people over the age of 45 are at a greater risk of heart failures and cardiovascular diseases such as atherosclerosis, hypertension, coronary and cerebral artery diseases.¹³ In these aging-associated pathological conditions, vascular cells appear to be unable to adapt to external stress and fail to maintain their normal quiescent phenotype, thus contributing to the onset of diseases.¹⁴ Growing evidence suggests that arterial elasticity and wall residual stresses decrease with age, which in turn may explain the prevalence of stroke and increased morbidity for cardiovascular disease among the elderly.^9,15

Aging is accompanied by the deterioration of the musculoskeletal system, including a decline in bone mass,¹⁰ a decrease in the size of muscle cells, fibers, and tissues, along with an increase in inelasticity and stiffness but a reduction in skeletal muscle strength.^16,17 With aging, particularly pathological aging, there is stiffening and fibrosis of the cartilage of joints that result in joint pain and restricted mobility.¹⁸ As a result, aging comes with a myriad of complications like muscle degradation,¹⁷ osteoarthritis,¹⁸ osteoporosis,¹⁹ and a general degradation of the capacity of the musculoskeletal system to accommodate the stress and strain of life.^16,20 For skin tissue, biomechanical degradation in the dermal fibroblasts and ECM environments contribute to the saggy appearance of aged skin and reduced protection against hazardous substances.¹¹ Aging is also associated with impairment of the human immune system and dysfunction in the adaptive and innate immune responses. Impaired immunity in aging has been attributed to delayed immune cell migration and inadequate crosstalk between antigen presenting cells and T-lymphocytes. Both are mechanotransductive processes that involve the dynamic remodeling of cytoskeleton and force transmission in immune cells.^21–23 The delayed and dysfunctional occurrence of mechanobiological processes in immune cells results in impaired immune response in the elderly. Thus, a clear understanding of the cellular mechanobiology of aging that primarily derives from studies on the dysregulation of cell mechanics, cytoskeletal architectures, signaling pathways, and forces that occur with normal and pathological aging would contribute to the development of therapies by intervening in the biophysical signals in the cell and the cell’s microenvironment.

The cellular mechanobiology of aging is an interdisciplinary research field involving numerous areas, from cell biology, disease pathology, biomedical technology, to computational modeling. This review aims to summarize the latest breakthroughs in aging-related cellular mechanobiology and provide insight into currently explored and unexplored mechanosensing and mechanotransduction mechanisms in the normal aging process and pathological aging-related disease development. This review will also highlight recent bioengineering approaches for revealing the key mechanobiological mechanisms that direct cell/tissue fate and function in healthy cells and cells in aging and related diseases.

Aging and cell mechanics

Aging is associated with prolonged changes in cellular mechanical properties (i.e., increased stiffness, decreased elasticity, and reduced strength) in the cardiovascular, musculoskeletal, skin, and immune systems. During the aging process, various types of tissue cells, including vascular smooth muscle cells (VSMCs),^24–28 epithelial cells,^29,30 cardiomyocytes,³¹ cardiac³² and skin^11,33 fibroblasts, osteocytes,³⁴ chondrocytes,^35,36 intervertebral discs,^37–39 tendons,^40,41 muscle cells,¹⁶ lymphocytes,¹² and red blood cells⁴² undergo a gradual cellular stiffening process that changes the mechanical properties and sensitivity of these cells. This ultimately leads to retarded cellular responsiveness and inferior functioning in aging tissues.² For example, aged human fibroblasts were found to migrate slowly, with shorter distance and lower directionality,² while the strength generation capacity of human muscle cells^16,20 and the contractile function of rat VSMCs²⁵ were weakened by aging. Eventually, these accumulating changes in the mechanical properties of aging cells leads to the malfunction of tissues and the human body since this collection of extensive abnormal cellular mechanics causes many aging-associated diseases.

Aging-associated cardiovascular diseases are major causes of death in the elderly. Different types of cells in the cardiovascular system undergo significant changes in cell mechanics during the aging process. VSMCs are the major cellular components of the vascular system. With aging, VSMC were found in various animal models to gradually stiffen because of increased collagen deposition and a decrease in elastin surrounding the vascular cells.^24–28 As a result, this aging-associated stiffening causes an attenuation of the ability of VSMCs to maintain the normal, frequent cyclic contraction and dilation in blood vessels and can trigger cardiovascular disease development.⁴³ Besides the vascular cells, aging in the heart is also associated with a number of structural and functional alterations in cell mechanics. For example, a significant increase in the elastic modulus of old mouse cardiomyocytes has been observed, which might contribute to the global changes in left ventricular diastolic dysfunction in aging hearts.³¹ Mouse cardiac fibroblasts acquire a profibrotic phenotype in the aged heart, which is a major cause of pathological interstitial fibrosis and ventricular stiffness in cardiac tissue.³² Cell adhesion, cytoplasmic viscosity, and cell spreading are all significantly increased during this process in such a profibrotic fibroblast, and this is accompanied by the enhanced crosslinking density of cardiac ECM and the severe hindrance of normal contraction in cardiac tissue.⁴⁴ The cellular structural and mechanical changes associated with aging thus cause cardiovascular diseases such as atherosclerosis,⁴⁵ atrial fibrillation,⁴⁶ heart failure,⁴⁷ hypertension,⁴⁸ intimal hyperplasia,⁴⁹ valvular disease,⁵⁰ and cardiomyocyte hypertrophy.⁵¹

Aging is associated with the deterioration of the musculoskeletal system, including a decline in bone mass¹⁰ and skeletal muscle strength,^16,17 and an increase in the stiffening and fibrosis of the cartilage of joints.¹⁸ In bone tissue, osteocytes are the most abundant and mechanoresponsive cells and play a key role in adaption to mechanical loading and the maintenance of bone mass by regulating both osteoblast and osteoclast activity.^34,52 Aging-related changes in osteocytes have been observed in mouse models, including increased cortical diameter, decreased cortical thickness, reduced density, and cortical porosities.^34,53 Such mechanical degeneration of osteocyte networks thus may lead to the loss of bone mass, delayed mechanosensitive calcium signaling, and impaired bone mechanosensitivity and responses to mechanical loading, which occurs in a gender-dependent manner, with females affected earlier and more severely than males.^34,54,55

Similar to the mechanical changes in the bone, there is also stiffening and fibrosis of the cartilage of joints that cause joint pain and reduced mobility through a pathological aging process in the elderly population.¹⁸ The elastic modulus of human chondrocytes have been found to increase significantly with osteoarthritis, an age-related degenerative disease in joint tissues.^35,36 Intervertebral disc degeneration is an age-related process and a major cause of spine stiffness, neck and back pain. Intervertebral discs are polyaxial cartilaginous joints consisting mostly of collagen ECM sparsely populated by fibrochondrocytic cells in the outer, fibrous annulus fibrosus ring and chondrocyte-like and notochordal cells in the central, gelatinous nucleus pulposus core.⁵⁶ The age-related degeneration significantly increases the compressive modulus of the outer anulus fibrosus^37,38 and shear modulus of the central nucleus pulposus³⁹ of human intervertebral discs. The increases in shear modulus suggests that the nucleus pulposus undergoes a transition from fluid-like behavior to more solid-like behavior with aging and degeneration.³⁹

Beside bone, cartilage, and intervertebral discs, aged tendons also undergo structural and biomechanical degenerative changes. Tendon fibroblasts, typically referred to as tenocytes, the main cellular component of tendons, show decreased density and cellularity but increased elongation with aging.⁵⁷ The viscoelastic properties and mechanical strength of mouse, rat, and equine tendons decrease with increasing age.^57–59 Aged human and mouse Achilles tendons were found to be significantly stiffer compared with young tendons.^40,41 However, contrasting evidence was also found in studies comparing tendon stiffness in older and younger adults, where the stiffness of human Achilles and patellar tendons decreased in older compared with younger adults.^60,61 Although there is not complete agreement on the mechanical changes that occur in tendon with age, age-dependent deterioration of tendon mechanics and function is no doubt a major risk factor for tendon injury, tendinopathy, tendonitis, chronic pain, tendon rupture, and limited mobility.

Muscle degradation and stiffening accompanying aging is another concern that plagues the elderly.¹⁷ A decrease in the size of muscle cells, fibers, and tissues, along with an increase in the inelasticity and stiffness and a reduction in skeletal muscle strength (force-generating capacity)^16,20 have been observed both in humans and mice during aging. All these features lead to an abnormally high tensile status in the cytoplasm and the decline of contraction in aged muscle cells. The aging-associated mechanical degradations in these musculoskeletal tissue cells thus lead to the risk of fractures, frailty,⁶² and complications like osteoarthritis,¹⁸ osteoporosis,¹⁹ and muscle degradation.¹⁷

Time-dependent changes in mechanical properties are an essential characteristic of the aging process of human skin. Mechanical measurements of individual dermal fibroblasts^11,33 and skin epithelial cells^29,30 isolated from human donors of different ages demonstrated that the stiffness of these human skin cells increases with age, which contributes to the age-related loss of elasticity in skin tissue.²⁹ The altered cell mechanics might involve changes in cytoskeleton and influence cell functions such as contractility, motility, and proliferation.^33,63 The aging of skin cells and the mechanical changes involved in the process are thus the keys to understanding skin aging.

Meanwhile, the obvious decline in essential immune function has been extensively observed in aged tissues, which may relate to changes in immune cell mechanics. Lymphocytes collected from older donors have been found to be slightly stiffer than young donors’ lymphocytes because of degeneration in the cytoskeleton.¹² Studies also revealed an association between membrane deformability and aging in human red blood cells that diminish the cell’s biological functions.⁴²

Collectively, the prolonged changes in cell mechanics of aged cells are largely associated with the progression of diseases. Although research suggests that age-dependent cell stiffening commonly occurs in the cardiovascular, musculoskeletal, skin, and immune systems, such an effect might not be uniform for all organs and tissues. Both stiffening and softening have been observed in different aging tendon tissues.^40,41,60,61 A few studies found no age-related stiffness difference in human pulmonary artery VSMCs in the lung⁶⁴ and mouse tendons,⁶⁵ and even a cell softening with age in some human skin fibroblasts.⁶⁶ Instead of changing cell stiffness, other mechanical property changes such as in ECM deposition and cellular traction forces were detected in these studies, which remains a topic that needs further exploration. Analyzing the altered cell mechanics of different aged tissue cells will facilitate our understanding of the mechanical machinery of these age-related diseases and boost the development of novel anti-aging therapeutic strategies.

Changes in ECM

It has been widely accepted that cellular mechanical properties are altered in the cardiovascular,^15,67,68 musculoskeletal,^34,69,70 and skin tissues^71,72 as a result of age-dependent changes to the composition and organization of the ECM. For example, aging-associated stiffening of VSMCs in rats is largely due to the increased collagen deposition and a decrease in elastin surrounding these vascular cells.^24,25 Data from rat models suggest that the ratio of collagen to elastin within the aortic wall increases with age and thus results in reduced vessel elasticity, as collagen is 100–1000 times stiffer than elastic fibers.^15,27 The aging‐mediated alteration in cardiac ECM composition and crosslinking affects cardiomyocyte function and mechanical properties, eventually leading to severe problems, including cardiac fibrosis and heart failure.^31,73

Aging-related mechanical degeneration of bone matrix networks may lead to the loss of bone mass and impair bone mechanosensitivity.³⁴ Osteocytes synthesize the mineralized bone matrix and form an extensive interconnected bone matrix network system (lacunocanalicular system) for nutrient exchange and the effective sensing of mechanical strain from the fluid flow in bones.⁷⁴ However, because of inferior matrix synthesis in aged osteocytes, the interconnectivity and mechanical stiffness of these matrix network system rapidly falls during aging.⁶⁹ The poorly interconnected bone matrix fails to support enough fluid strain transmission to the osteocytes in aged bones, leading to decreased bone mechanosensitivity and force generation in osteocytes.⁷⁵ In cartilage, oxidative stress induces a reduction of the synthesis of ECM components by chondrocytes, leading to an alteration of cartilage structure and the subsequent decline of the tissue’s mechanical properties, with the appearance of fissures, fragmentation, and joint diseases such as osteoarthritis.⁷⁰

Fibroblasts are extensively present in the skin and the interstitial spaces of tissues and produce large amounts of ECM to support other functional cells in the tissues. The age-induced mechanical changes in human dermal fibroblast cells are associated with the changes in collagen and elastin organization and density of the ECM.^11,71 Alterations in matrix metallopeptidases (MMPs)^63,72 and the cross-linking and viscoelastic properties of the collagen matrix^71,76 lead to the loss of support and mechanical cues in the aging dermal fibroblasts. The degradation in the mechanical properties of dermal fibroblasts and digested ECM environments contribute to the saggy appearance of aged skin and reduced protection against hazardous substances. Moreover, a critical aspect of repair in young skin is the outgrowth of keratinocytes from eccrine sweat glands (ESGs), and aging is associated with a decrease in the formation of these outgrowths, leading to a loss of skin repair with aging.⁷⁷ Failure to form cohesive ESG outgrowths in old human skin may be attributed to impaired interactions of keratinocytes with the skin dermal ECM because of age-associated damage, which includes increased collagen fiber fragmentation, reduced ECM resistance, and decreased tissue mechanical force.⁷⁷

Changes in cytoskeleton

The cytoskeleton is made up of actin filaments, microtubules, and intermediate filaments and is responsible for maintaining cell morphology, organization, and critical cellular functions like movement and division.⁷⁸ Abnormalities in cytoskeleton remodeling have been reported as the key drivers of the changing cell mechanics and malfunction during cellular aging (Fig. 2). The actin filament network provides mechanical support to the cell and is involved in important cellular processes such as migration, division, cell shape regulation, contractility, and mechanosensation.⁷⁹ Aging has been associated with changes in actin filament structure and function in different types of tissue cells.⁸⁰ For example, human VSMCs⁶³, ECs,⁸¹ and dermal fibroblasts³³ show a significant increase in actin filament polymerization with age, leading to a highly condensed and stiffened cytoskeletal network, with a large overall area, high stiffness, and low motility. In hypertension, which commonly happens in the older population, the highly polymerized actomyosin networks in VSMCs and ECs lead to the abnormal contraction of the cells and the inward remodeling of the blood vessel.^81,82 Cytoskeletal reorganization also likely plays a key role in human trabecular meshwork cell stiffening and glaucoma disease development in the elderly population, whereas altered F-actin morphology is associated with trabecular meshwork cell dysfunction.⁸³ Early work on the cytoskeleton of human peripheral blood lymphocytes suggests that the concentration of F-actin fibers increases in older cells, but stimulus-induced actin polymerization was found slightly to be lower in older lymphocytes.¹² As a result, a slight increase in the cell stiffness can be expected for older lymphocytes compared with young cells. Studies also show that aging alters the F-actin in CD4⁺ T cells, which causes declines in T cell activation, membrane fluidity, and defects in the formation of the immunological synapse.^21–23 In addition to lymphocytes, aging-impaired F-actin polymerization was found to reduce alveolar macrophage phagocytosis in a mouse model.⁸⁴ The decreased macrophage-dependent immune functions and accumulated apoptotic debris could contribute to immune system dysfunctions that occur in aged organisms and chronic inflammation.⁸⁵

Figure 2.

Critical mechanobiological cellular components affected by the aging process.

Like actin filaments, the primary role of the microtubules in cytoskeleton is mechanical and their structure and function may undergo age-related changes that result in cellular dysfunction and age-associated diseases. Early research revealed that centrosomes and the microtubule network become significantly affected in aging Drosophila cells.⁸⁶ The declines in total and polymerized tubulin, together with the increases in the free fractions of microtubule-associated proteins (MAPs) indicate fewer and/or shorter microtubules in old rats.⁸⁷ Microtubules are highly dynamic and the mechanical properties of microtubules are very important for their function.⁸⁸ Both experimental and modeling studies demonstrated that aging microtubules have a higher catastrophe rate for switching from assembly to shortening than younger microtubules.^89,90 The aging-accompanied alterations in the actin network also have important regulatory effects on microtubules.⁹¹ As microtubules mainly function in the maintenance of cell-shape by bearing tension, particularly compression, weakening of the microtubule network in aging affects maintenance of cell shape and function, for example, cell division, motility, secretion, and ligand-receptor endocytosis. Thus, pathological aging-accompanied perturbations of microtubule functions can lead to diseases such as Alzheimer’s, muscular atrophy, cardiac dysfunction, and cancer.^86,87,92

Apart from actin filaments and microtubules, intermediate filaments serve as the most durable component responsible for stabilizing cellular organelles from the effects of mechanically induced stress. Overexpression of intermediate filaments was observed in human dermal fibroblasts derived from the old donors compared with those from young and newborn donors.⁹³ Aging-associated remodeling of intermediate filaments has also been found to affect the changing viscoelastic properties of human dermal fibroblasts⁹³ and chondrocytes³⁵ in the joint tissue affected by osteoarthritis. Overexpressed intermediate filaments are extensively involved in over 45 kinds of chronic diseases caused by aging.⁹⁴ For example, changes in intermediate filaments in the cardiovascular system lead to dilated cardiomyopathy, which is the most common cause of heart failure in clinical settings.⁹⁴

Changes in nuclear mechanics

As the nucleus is directly linked to the highly dynamic cytoskeleton, mechanical signals generated within this network and from the extracellular microenvironment will converge on the nucleus and directly impact its morphology and overall function. Nuclear mechanics is thus crucial in protecting DNA from damage and mediating nuclear mechanotransduction in response to cell-intrinsic and -extrinsic mechanical forces. Nuclear mechanics is mainly determined by two major mechanical components: the rigid lamin filaments forming a thin meshwork at the periphery and the viscoelastic chromatin filling the interior of the nucleus.⁹⁵ Nuclear lamins, particularly lamin A filaments, which are specialized type V intermediate filaments, have been reported to internally determine nuclear mechanics⁹⁶ and serve as an indicator of aging cells.⁹⁷ Similar to the ECM surrounding the cell, nuclear lamin A surrounding the nuclear membrane provides the connection between the nuclear inner membrane with the chromatin to stabilize the nucleus in the cytoplasm, and determines the shape, size, and mechanical properties of the nucleus. Remarkably, the stiffness of the nucleus increases significantly in progeria and old human dermal fibroblasts because of aberrantly high expression of mutant lamin A.⁹⁸ The lamin A filament networks provide extensive mechanical support of the nucleus and limit the potential deformation of the nucleus in response to mechanical signals, and irregular deformation and significantly higher stiffness of the nucleus is observed in premature aging cells compared with normal cells.⁹⁸ These damaging changes in the nucleus ultimately influence gene expression and modulation of overall cell mechanics in aging cells. A critical pathological aging-related disease is Hutchinson-Gilford progeria syndrome, where mutant lamin A filaments lead to an increase in nuclear stiffness and a more brittle and solid-like nucleus,⁹⁹ premature aging,¹⁰⁰ and potential apoptosis¹⁰¹ in affected cells. A study by Scaffidi et al. showed that inhibition of an laminA aberrant splicing event can reverse aging-associated defects in nuclear structure, showing the close association between the nuclear deformities and lamin A and providing a possible therapeutic approach to rescuing nuclear degradation caused by aging.⁹⁷ Mechanosensitive lamin A expression and nuclear stiffness has been found to be affected by ECM stiffness.^96,102 Nuclear architecture and pathological cell senescence can be regulated by an ECM-remodeling membrane protease in a process involving the regulation of nuclear stiffness by the ECM under cell stress conditions.¹⁰² Understanding these nuclear mechanics-based mechanisms is thus beginning to affect therapeutic approaches to aging-related diseases.

Besides lamins, chromatin is another major mechanical component that dominates the mechanical resistance of the nucleus to small deformations.⁹⁵ Chromatin filling the nucleus provides mechanical support for the nucleus to resist forces, maintain nuclear mechanical stability, and protect DNA from damage via compartmentalization maintenance. Alterations in chromatin-mediated nuclear mechanics and downstream loss of nuclear shape and stability have been shown to result in nuclear rupture and dysfunction and increased DNA damage.¹⁰³ Significant chromatin structural changes and DNA damage commonly occur in the nucleus during physiological aging and senescence. In the mutant lamin A–induced Hutchinson-Gilford progeria syndrome premature aging disease, cell nuclei show changes in internal chromatin organization, loss of heterochromatin condensation, and accumulation of DNA damage^100,103 Research also suggests that chromatin decompaction treatments show a potential to rescue Hutchinson-Gilford progeria syndrome by changes to nuclear rigidity without requiring alterations to lamins.¹⁰³ Despite the clear biological effect of chromatin alterations on the aging process, the current understanding of the causes and outcomes of these nuclear mechanics changes in aging remains limited. The ability to differentiate the contribution of chromatin and lamins of the nucleus, the two major mechanical components, will provide unprecedented understanding of aging process and aging-related diseases. It will require novel tools for more precise measurements of the dynamic mechanical properties of the nucleus and subnuclear structures, as well as targeted mechanical stimulation with high spatiotemporal resolution and live-imaging capabilities.⁹⁵

Altered cellular force in aging

The various types of mechanical forces (e.g. tension, contraction, compression, and shear stress) in the cell and its environment are essential for many biological processes, from molecular activation,¹⁰⁴ cell adhesion¹⁰⁵, migration,¹⁰⁶ and stem cell differentiation¹⁰⁷ to tissue morphogenesis,¹⁰⁸ organ development,¹⁰⁹ wound healing,¹¹⁰ and even cancer progression.¹¹¹ Accumulating evidence has correlated aging with progressive changes of mechanical forces in a wide range of tissue cells due to aging-associated degeneration of the ECM⁶³ and progressive inflammation⁶⁸ in the tissue environment as well as the dysregulation of the intrinsic cytoskeleton and mechanosensitive signaling in the cell.²

During the aging process, cellular forces in the cardiovascular, musculoskeletal, and skin tissues are largely affected by degraded ECM. For instance, due to the lower expression of collagen protein and less organized collagen fibers, aged mouse and equine tendons demonstrate significantly less tensile strength in response to an induced strain.^57,58 The traction force of human dermal fibroblasts⁶³ is largely decreased during aging because of poorly interconnected supporting ECM, while the overexpression of cardiac ECM and vinculin induces higher contractile force in aged cardiomyocytes in animal and Drosophila models.⁶⁷ Progressive inflammation is another major factor that increases the tensile force in aging cells. In aged mouse cardiovascular system, key characteristic inflammatory markers, including B cell lymphoma 6 (BCL6), C-C motif chemokine ligand 24 (CCL24), interleukin 4 (IL-4), and MMP-9, are highly expressed by cardiomyocytes.⁶⁸ The upregulated inflammatory status of the aged murine cardiomyocytes stimulates the overexpression of the cardiac ECM, induces higher tension force in aged cardiomyocytes, which causes abnormal thickness and reduced diastolic function in the left ventricle.^67,68 The progressive inflammation and high tensile force status also cause aging-related fibrotic tissues in the respiratory system. In pathological aging-related fibrotic diseases such as idiopathic pulmonary fibrosis, the cellular traction force is elevated due to progressive inflammation in the aging cells.^112,113 Moreover, Schafer et al. found that treatment with aged cell–conditioned media containing inflammatory cytokines can elevate the traction force of normal lung fibroblasts.¹¹² Collectively, these results clearly show the effects of degraded ECM and prolonged low-grade inflammation in the tissue environment on cellular tension force in aging.

Apart from the influence of the extracellular environment, aging-associated degeneration of the cytoskeleton and mechanosensitive signaling also regulate cellular tension force. This dysregulation of cytoskeletal actin filaments, microtubules, and intermediate filaments is responsible for retarded mechanosensitivity and response to external stimuli, which leads to impaired cellular functions with age. The in vitro measured traction forces of human dermal fibroblasts and ECs increase with age and are associated with increasing F-actin content, bundling, and localization in the cell.² Prolonged high cellular tension in pathological aging may eventually cause hypertension, stroke, aneurysms, atherosclerosis, and heart disease. Owing to the changes in the cytoskeleton, such as a more diffused actin filament network, diabetic VSMCs showed weaker force response to the external mechanical stimuli.¹¹⁴ Thus, the novel concept of cellular allostasis, explored in the context of differentiating between the force dynamics of healthy and diseased VSMCs, could very well be applied to understanding the functional decline with age of the response to mechanical stimuli.¹¹⁵ Similarly, aging chondrocytes are no longer responsive to cyclic loading for enhanced biosynthesis because of highly polymerized cytoskeleton networks.^116,117 In skeletal muscle, the molecular motor myosin generates force and movement. An aging-related slowing in the speed of actin filaments propelled by myosin has been observed at the whole muscle and single fiber levels in mice, rats, and humans.¹¹⁸ Due to the age-related changes in myosin, the contractility of single muscle fibers of old rats is significantly lower than that of fibers from young rats, suggesting age-related decline in muscle function.¹¹⁹ The loss of muscle force and contractile speed in the elderly results in sarcopenia and greater risk for falls and fall-related injuries.¹¹⁸ As the intermediate filament remodeling rate of human dermal fibroblast decreases with donor age, aged human dermal cells show a lack of ability to adapt to mechanical stress.⁹³ A decline in cytoskeleton remodeling rate was also observed in aged mesenchymal stem cells.¹²⁰ Such a decline reduces cellular mechanosensitivity and the reactions to mechanical stimuli, which finally hampers the motility of aged cells and accelerates the cells to undergo senescence and apoptosis.

The generation of cellular force also relies on cell-to-cell interactions, which are also dysregulated during aging yet remain largely unstudied. A change in the intercellular mechanical interactions would affect many important biological processes, including collective cell migration and alignment in wound healing and tissue morphogenesis.^121,122 Intercellular forces and the digestion of tight junctions also determine the local tension force in tissue cells and thus influence the mechanical elimination of aging cells.¹²³ The development of novel high-resolution force measurement platforms that enable monitoring the spatiotemporal dynamics of intracellular and intercellular forces will greatly assist the discovery of force-related mechanisms in multicellular organisms and the understanding of homeostasis in various aging tissues.

Aging-related mechanotransductive signaling

It has become increasingly clear that cellular behaviors in the aging process are not only regulated by extrinsic soluble biochemical factors and intrinsic biological signals but also by biophysical cues in the cellular and extracellular microenvironment through the process of mechanotransduction.⁸ The cellular mechanotransduction signaling process, involving cell adhesion, cytoskeleton, and nuclear-related signals, is significantly affected during aging (Fig. 3).^8,124 Mechanical signal sensing and transduction in old cells becomes less effective than that in young cells, thus dysregulating the basic biological responses and functionality of the cell.

Figure 3.

Major signaling pathways involved in aging-regulated cell mechanobiology.

Changing mechanical cues in the ECM first have to be sensed and translated into the biochemical signals through the dynamic formation of focal adhesions (FAs).¹²⁵ One of the earliest events during FA formation and mechanosensing is the activation of integrin receptors and integrin clustering. Integrins bind to the ECM and can sense and transduce mechanical signals, such as the ECM rigidity, composition, nanoscale ligand architectures, and forces, into the cytoplasm through the mechanosensitive FA proteins and focal adhesion kinase (FAK).^105,126 Essential FA proteins, including paxillin, vinculin, talin, and FAK, are overexpressed in the perinuclear region of aged cells instead of the lamellipodium region, as in normal cells,^127–129 showing that both the organization and activation of FA proteins is affected by aging. Immunoblot analysis of the aorta of adult and old rats determined that the aortic content of FAK, FAK-related non-kinase (p41-FRNK), Src, and the phosphorylation status of FAK and paxillin all showed an increase with age.¹²⁸ Aged rat VSMCs and human diploid fibroblasts show a hyperadhesive phenotype, with extensive clustering of integrins, large FAs, and increased expression and phosphorylation of FAK.^24,127 Further, aging-regulated FA signaling in human diploid fibroblasts is accompanied by an increase in mechanosensitive activities of Rho GTPases, Rac1, caveolin-1, and cell division cycle 42 (CDC42).¹²⁷ Thus, these changes in FA mechanotransduction are translated into mechanical dysregulation of the cellular cytoskeleton and ultimately result in age-associated malfunction and diseases. Promising results have shown the possibility of rescuing the age-related changes in FA mechanotransduction, where the knock-out of caveolin-1 in old fibroblasts can inactivate FAK, disrupt FA formation and actin stress fibers, and cause a change in the cell shape towards a small spindle shape that is normally associated with young cells.¹²⁷

Besides mechanosensing through integrin and FA, many downstream mechanosensitive signaling pathways, such as the Hippo/YAP (Yes-associated protein) signaling pathway, are dysregulated in the aging process.¹³⁰ Important for development, stem cell differentiation, organ size control, and cancer, the Hippo/YAP pathway has recently been identified as a convergent signaling pathway for cytoskeleton mechanics and a critical nuclear-related mechanotransduction pathway.¹⁰⁷ In Hippo/YAP signaling, YAP and TAZ (transcriptional coactivator with PDZ-binding motif) are the two key Hippo transducers for effective transduction of mechanical signals from the ECM through FAs and from the cytoskeleton to the nucleus, and for triggering cellular responses.¹³¹ Mechanical cues such as ECM rigidity and nanotopography can be sensed by the cell and transduced through the F-actin cytoskeleton to suppress the phosphorylation activity and nuclear localization of YAP/TAZ,^107,126 which induces downstream signaling activation in the nucleus.¹³² Aging alters the ability of YAP and TAZ to transduce mechanical information to the nucleus in human mammary epithelial cells.¹³³ In aged human mammary epithelial cells, dermal fibroblasts, and adipose ECs, YAP/TAZ nuclear localization activity is decreased while the inhibitory phosphorylation of YAP/TAZ is increased in the cytoplasm.^93,133,134 When YAP is predominantly localized in the cytoplasm of these aged cells, the transcriptional activity of its target genes as well as the self-repair and self-renewal capability of the cell significantly decrease with age. However, in another study, owing to altered ECM and cell mechanics, aged mouse muscle fibroblasts isolated from a stiff microenvironment display increased nuclear levels of the mechanosensors YAP/TAZ.¹³⁵ The discrepancy of YAP/TAZ nuclear localization in different types of tissue cells may result from the different cell spreading areas associated with age, which is a topic that needs to be further studied.⁹³ Age-dependent changes in cell size and/or YAP activity may serve as the key control point of age-related decline in angiogenesis. Indeed, it has been shown that the reduction of aged human EC size by micropatterning cells or the stimulation of YAP activity in aged ECs can attenuate EC senescence and restore blood vessel formation.¹³⁴

Apart from the Hippo/YAP pathway that declines in the aging process, some other mechanosensitive signaling pathways are upregulated and accelerate the progression of senescence in aging cells. The Hippo/YAP pathway has been found to be regulated by intrinsic RhoA (Ras homolog family member A)/Rho-associated protein kinase (ROCK)-mediated cytoskeleton tension.^107,126 Likewise, overactivated RhoA signaling in aged mouse muscle cells causes abnormal high tensile and stiffening of the cell,¹³⁶ which may be accompanied by dysfunction in Hippo/YAP pathway that needs yet to be further explored. Mitogen-activated protein kinase (MAPK) has been proven to be important in mechanosensitive signaling and plays important roles in cell proliferation, differentiation, and apoptosis. Mechanical stimulation can activate MAPK signaling in different tissue cells, such as human cartilage,¹³⁷ epithelial cells,¹³⁸ mouse fibroblasts,¹³⁹ rat cardiomyocytes,¹⁴⁰ and cardiac fibroblasts.¹⁴¹ Recent work suggests that mechanical stress activates p38 MAPK and induces cardiac hypertrophy through the integrin-FAK-Src-Ras pathway in rat cardiaomyocytes.¹⁴⁰ As the integrin-FAK-Src-Ras pathway has been found to increase with age,¹²⁸ MAPK activation might also be enhanced during the aging process. For example, p38 MAPK is more activated in human granulosa cells,¹⁴² macrophages,¹⁴³ and rat aorta¹⁴⁴ from older subjects than in those from younger subjects. However, compared with the higher resting phosphorylation state of p38 MAPK, acute exercise or mechanical loading-induced phosphorylation of p38 MAPK was found to be attenuated in aged rat aorta¹⁴⁴ and human skeletal muscle cells.¹⁴⁵ Thus, aging alters the ability of muscle and vessels to initiate intracellular signaling appropriately in response to mechanical load. These observations indicate that MAPK signaling is regulated mechanically and that control of its activation is changed with aging. Inhibiting MAPK signaling in elderly individuals may rejuvenate their response to be more similar to that of younger people. Akt is a another highly mechanosensitive signaling pathway, and its activation is thought to be mediated by muscle loading and correlated with the changes in muscle mass with age.^146,147 The mammalian target of rapamycin (mTOR) signaling pathway is regarded as a major aging-associated downstream signaling pathway of Akt that leads to various malfunctions, including dysregulated mechanosensing and altered intercellular communication.^147,148 The mTOR-FAK mechanotransduction signaling axis is critical for FA maturation and cell proliferation,¹⁴⁹ which might be significantly affected in the aging process.

Accompanied by reduced mechanotransduction, the inflammation in aged cells is commonly intense.¹⁵⁰ The robust activation of mTOR in aged cells represents a transition of human fibroblasts and breast epithelial cells from the mechanosensing phenotype into the secretory phenotype.¹⁵¹ By inducing the aged cells to secrete senescence-associated cytokines, a large portion of these cells undergo inflammation, degradation, and even programmed apoptosis. Hence, the mTOR pathway has been implicated in the etiology of a number of pathological aging-related diseases, such as Alzheimer’s disease, cancer, type 2 diabetes, kidney, heart, and autoimmune diseases, and decline in cognition and immune system.¹⁵² Thus, inhibition of mTOR signaling is a promising therapeutic approach to improve the inflammatory microenvironment in the aged tissues and treat pathological aging-related diseases.¹⁵¹ Inflammation in aged cells is directly caused by oxidative stress and reactive oxygenation species (ROS).¹⁵³ ROS rapidly trigger the expression of various harmful inflammatory cytokines, including TNF-α, IL-1α, and IL-6, which induce the loss of cell polarization, cell-cell adhesions, and specialized cell functions.¹⁵⁴ Excessive inducible nitric oxide synthase (iNOS) is a biomarker for the elevated inflammation of aged cells,¹⁵⁵ and the abnormal iNOS-mediated nitric oxide (NO) pathway has been linked to poor mechanosensation, motility, and proliferation in mouse ECs.¹⁵⁶ Modulating the inflammatory signaling in aged cells thus might be an effective approach to improve cellular mechanosensing and mechanotransduction.

Collectively, an understanding of the essential roles of these mechanotransductive signaling pathways will provide valuable insights into the molecular mechanisms behind normal and pathological aging-related processes and diseases. More studies are needed to explore other mechanotransductive signaling pathways associated with aging in different types of cells and involving a more dynamic and functional quantification of the cellular mechanobiology. This would facilitate our understanding of the natural aging process along with providing valuable biophysical markers for the diagnosis and treatment of pathological aging-related diseases.

Current bioengineering strategies for deciphering cellular mechanobiology in aging

The recent development of novel bioengineering approaches is essential for studying the key mechanobiological mechanisms in aging. Emerging single-cell and single-molecule technologies have been developed and applied to measure the changes in mechanobiological characteristics in aging cells, including forces, cytoskeleton architectures, and mechanosensation (Fig. 4).

Figure 4.

Current bioengineering strategies for deciphering cellular mechanobiology in aging. (A) Atomic force microscopy. (B) Förster resonance energy transfer (FRET) sensor. (C) Single cell traction force microscopy. (D) Elastic micropillar arrays. (E) Monolayer stress microscopy for multicellular force analysis. (F) Microtweezers systems for probing cell mechanics.

Atomic force microscopy for measuring cell mechanics

Atomic force microscopy (AFM) is one of the major techniques used for the measurement of cell mechanics during the aging process (Fig. 4A). AFM uses a vertical microcantilever to measure the stiffness and force of the cell to study the correlation between the changing cellular mechanics and aging.^{11,29,31,63,157} Due to its extremely high detection resolution, AFM can map subcellular mechanical properties and measure the different rigidities of the nucleus, cytoplasm, and cell edge.^29,98 AFM measurement allows for quantitation of the cellular cytoskeleton and demonstrates that older human epithelial cells have a denser and thicker cytoskeleton than younger cells.²⁹ In addition to cellular analysis, AFM is also a highly sensitive technique to measure the organismal biomechanical properties of aging Caenorhabditis elegans and Drosophila.^67,157 In vivo AFM mechanical measurement revealed mechanical degradation associated with aging, and cuticle senescence and stiffness can be used as biomarkers of ageing and health span. Overall, AFM has been extensively applied in aging mechanobiology research and has discovered various age-associated changes in cell mechanical properties.^31,157,158

Single-molecule Förster resonance energy transfer microscopy (FRET) for visualizing the activation of mechanotransduction signaling

FRET-based molecular sensors have been widely used for dynamic monitoring of mechanosensitive molecular activities in the cell (Fig. 4B).^159,160 In FRET measurements, the extent of nonradiative energy transfer between two genetically encoded fluorescent dye molecules, referred as a donor and an acceptor, reports the molecular activities in the cell.¹⁶¹ Aging has often deleterious effects on the mechanosensitive signaling pathways inside the body that can be measured with FRET probes. For example, the genetically encoded pRaichu-RhoA FRET mechanosensor has been developed to monitor RhoA activity in fibroblasts upon cyclic stretching.¹⁶² In another study, a FRET sensor was used to track the spatiotemporal activation of Src in human ECs in response to an external force stimulus applied to the cell membrane, revealing the directional mechanotransduction of force through Src signaling in the cell.¹⁶³ Similarly, mechanosensitive Rac activation was also monitored with FRET in bovine aorta ECs in response to shear stress.¹⁶⁴ Calcium FRET biosensors are commonly used to study calcium oscillations and the mechanical responses of cells to external mechanical cues such as force stimulation and substrate rigidity.¹⁶⁵ In addition to the study of mechanosensitive signaling, an actinin-sstFRET mechanosensor has been used to monitor subcellular tension force in real-time.¹⁶⁶ All these FRET-based mechanosensors thus are particularly useful tools for studying mechanosensitive signaling and mechanotransduction mechanisms in aging.

Traction force microscopy for force analysis in aging cells and multicellular organisms

Cellular traction forces are determinative for cell functionality and influence the migration, proliferation, development, and differentiation of cells in aging. Traction force microscopy (TFM), based on a fluorescent bead–embedded hydrogel substrate¹⁶⁷ or elastic polydimethylsiloxane (PDMS) micropillar array,^114,168 has been commonly used to measure cellular traction forces. For example, polyacrylamide hydrogel–based TFM (Fig. 4C) has been used to determine age-associated changes in traction forces exerted by aging ECs,⁸¹ fibroblasts,¹⁶⁹ and VSMCs.¹⁷⁰ Hydrogel-based TFM uses fluorescent microbeads–embedded polyacrylamide hydrogel as a cell culture substrate. The traction forces generated by individual cells can be obtained by comparing two sets of fluorescent images of the embedded fluorescent microbeads underneath the cells before and after removing the adherent cells. The displacement of the fluorescent beads underneath the cells indicates the substrate deflection under the traction force, which can then be used to calculate the stress exerted by the cells through analytical techniques such as Fourier transform traction cytometry (FTTC) or the finite element approach.¹⁷¹ Elastic PDMS micropillar arrays are another common force measurement technique used in cellular mechanobiology studies (Fig. 4D).^{105,114,168,172} When cells are cultured on PDMS micropillar arrays, the cellular contractile forces bind the micropillars underneath the cells. The deflection of the micropillars can be recorded with fluorescent dyes and live cell imaging to calculate the force exerted by the cells. Elastic PDMS micropillar arrays have been used to measure cell traction forces exerted by young and aged cells.¹⁷³ These TFM studies have successfully shown that cellular senescence has deteriorating effects on the force generation capacity of the cells.

In addition to force measurement, both the PDMS micropillar array and fluorescent bead-embedded hydrogel TFM substrate can serve as a mechanically tunable substrate to mimic the changing rigidity of ECM during aging. The effective elastic moduli of the PDMS micropillar array and polyacrylamide hydrogel-based TFM substrates can be modulated by changing the geometry of the micropillars (i.e., pillar height, diameter, and center-to-center distance)^107,111 and the chemical compositions of the hydrogels,¹⁷⁴ respectively. Such an approach is highly compatible with aging research, since aging is often associated with uniform and macroscopic changes in the ECM, as shown by studies done on ECs and fibroblasts.^135,173 Consequently, TFM with tunable rigidity has been used in studies on age-related intimal stiffening, which is characterized by a non-uniform point-to-point heterogeneity in sub-endothelial matrix stiffness.^173,175

TFM also serves as the foundation of many inferential methods for the determination of cellular stress and intercellular forces in multicellular systems. One of the basic tools used in such investigation is monolayer stress microscopy¹⁷⁶ (Fig. 4E), which has led to a plethora of new discoveries regarding the role of stresses and forces in regulating cellular behavior. Analytical frameworks have been created to map intercellular forces and have provided insights into the role of cadherin junctions in directing intercellular forces¹⁷⁷ and the effect of intercellular forces in guiding local cellular migration in the cellular monolayer.^122,178 Aging has been associated with reduced mobility of cells and reduced wound healing rates. An investigation of the changes in the nature of forces in healthy and old cells can help in developing strategies that can counter the deleterious effects of aging and possibly pave a way forward to reverse it.

Microtweezers systems for probing cell mechanical responses

Besides the direct measurement of cellular forces, another important aspect of mechanobiology in aging is to understand the mechanical responses of cells to mechanical stimuli. Innovative cellular micro-tweezers systems (Fig. 4F), such as magnetic tweezers,¹⁷⁹ optical tweezers,¹⁸⁰ and ultrasound tweezers,¹¹⁴ have been developed to apply mechanical forces to cell. The underlying principles of these microtweezers techniques are distinct, but these tools commonly apply forces, such as a magnetic field, laser beam, or ultrasound waves, to small particles bound to the cell surface. For example, magnetic tweezers apply a magnetic field to manipulate magnetic particles attached to the cell surface.¹⁷⁹ Optical tweezers cast a highly focused laser beam to trap and manipulate microscopic and neutral objects such as the small dielectric spherical particles on the cell surface.¹⁸⁰ Ultrasound tweezers utilize ultrasound waves to extensively manipulate microbubbles bound to the cell via integrin-arginine-glycine-aspartate (RGD) binding to apply forces to the cell membrane.^114,172 Specifically, by stretching the magnetic beads bound to the VSMC membrane through the RGD peptide, magnetic tweezers–based measurement can determine the stiffness of cell and tissue based on the ratio of force applied on the cell membrane to the displacement of the magnetic beads.¹⁸¹ Another study using a similar magnetic tweezers system determined the force-mediated cytoskeletal stiffening and its associated mechanotransduction response via the integrin-RhoA pathway.¹⁸² Also, magnetic tweezers can internally apply a mechanical force to cell through a magnetic particle ingested by the cell, allowing to determine how cytoskeleton filaments act in unison to maintain cellular integrity.¹⁸³ Similarly, optical tweezers have been used to apply mechanical forces to the cell membrane and showed that the high strength and duration of the pulses would induce more cellular calcium transients in the mouse neuroblastoma cells.¹⁸⁴ Integrated with TFM and FRET mechanosensors, ultrasound tweezers have been used to study cellular allostasis behaviors in responding to external force stimulation to differentiate between healthy and diseased VSMCs.^114,166 Thus, these integrated microtweezers systems can be readily utilized in the studies on cell mechanobiology in aging to decipher the changing mechanical behaviors of tissue cells that lead to functional deterioration in the aging cells and aged tissues.

Conclusions

Aging is associated with various mechanobiological changes in cells and results in different biological dysfunctions and age-related diseases (Table 1). In this process, cellular mechanics and ECM commonly undergo measurable changes and the cell tends to become stiffer with age overall. All components of the cellular cytoskeleton, including the actomyosin assemblies, microtubules, and related motor proteins and intermediate filaments, are altered in aged cells. Since cellular mechanotransduction depends upon a healthy and functioning cytoskeleton, the dysregulation of the cellular cytoskeleton leads to dysregulation of a cascade of mechanobiological signaling pathways, impaired cell force activities, and impaired functions. These major mechano-pathways, such as integrin/FA adhesion signaling, contractile-related RhoA/ROCK pathway, Hippo/YAP, MAPK, and Akt/mTor pathways, nuclear lamin-A and chromatin, as well as ROS-mediated inflammatory signaling, are key mediators and potential therapeutic targets for aging and related diseases. The dysfunction of these critical mechanobiological features in the cell and the ECM environment during the aging process influences mechanical force sensing and transmission in the cell. As a result, it dysregulates various biological processes, including adhesion, migration, gene expression, and cell fate, that are vital for the maintenance of tissue homeostasis. Therefore, a clear understanding of these changes in cellular mechanics, cytoskeleton structures, forces, and mechanotransduction pathways can help in discovering novel biomarkers for aging and new approaches to restore the defects caused by aging.

Table 1.

Aging-associated mechanobiological alterations in various cell types and related diseases.

Organs	Cell type	Mechanobiological changes	Age-associated diseases	References
Cardiovascular system	Vascular smooth muscle cells	Stiffness ↑ tension force ↑ ECM collagen↑, elastin ↓ adhesion ↑ actin polymerization ↑ YAP phosphorylation ↓	Hypertension, stroke, angina	24–28,63,64,82
	Endothelial cells	fibrotic ECM ↑ YAP phosphorylation ↑ inflammation ↑	Atherosclerosis	45,81,134
	Cardiomyocytes	Stiffness ↑ cardiac ECM ↑ microtubule activities ↓ intermediate filaments ↑ tension force ↑ inflammation ↑	Hypertension, cardiomyocyte hypertrophy, heart failure, valvular disease, myocardial infarction, cardiomyopathy, cardiac fibrosis, interstitial fibrosis	31,50,51,67,68,92,93
	Cardiac fibroblasts	Stiffness ↑ adhesion ↑ cardiac ECM crosslinking ↑ tension force ↑ inflammation ↑	Heart failure, idiopathic pulmonary fibrosis, atrial fibrillation	32,44,46
Musculoskeletal system	Myocytes	Stiffness ↑ fiber length ↓ muscle strength ↓ RhoA signaling ↑ tension force ↑ adhesion ↑ Akt/mTOR ↑ p38 MAPK ↓	Sarcopenia muscle degradation	16,17,136,145
	Osteocytes	Stiffness ↑ tension force ↓ calcium influx ↓ bone matrix network ↓ mechanosensitivity ↓	Osteoporosis, bone fractures	19,34,52,55,69,75
	Chondrocytes	Stiffness ↑ ECM synthesis ↓ intermediate filaments ↑ actin polymerization ↑ YAP phosphorylation ↑ mechanosensitivity ↓	Osteoarthritis, joint pain, cartilage fibrosis	18,35,70,116,117
Skin	Dermal fibroblasts	Stiffness ↑ adhesion ↓ actin polymerization ↑ intermediate filaments ↑ Lamin A mutation ↑ collagen crosslinking ↑ tension forces ↓	Diminished skin repair, saggy skin, Hutchinson-Gilford progeria syndrome	11,63,71,72,76,77,93,98
	Skin epithelial cells	Stiffness ↑ ECM rigidity ↑	Alzheimer’s disease, cardiomyopathies	29,30
Immune system	Lymphocytes	Stiffness ↑ F-actin concentration ↑ stimulus-induced actin polymerization ↓	autoimmune diseases, immune deficiency	12,21–23
	Macrophages	Stiffness ↑ actin polymerization ↓ p38 MAPK ↑	Wiskott-Aldrich syndrome, immune deficiency	84,143
	Red blood cell	Stiffness ↑	immune deficiency	42
Eye	Trabecular meshwork cells	Stiffness ↑ F-actin ↑ intermediate filament ↑	Glaucoma	83

Since aging is reflected by the malfunction of a variety of organ systems, functional mechanobiological studies on the variety of changes in different types of tissues and organs are an urgent need. Even in the same organ or tissue system, cell-to-cell variation may play an important role in the aging process. Plenty of evidence has revealed cellular heterogeneity in aging-associated mechanobiological changes. Cells can either undergo stiffening, softening, or no significant change,^40,41,60,61 and cellular forces and mechanotransduction signaling such as YAP^93,135 and MAPK¹⁴⁴ are either overactivated or diminished due to different ECM alterations and/or cytoskeletal remodeling in the aging process.^2,118 In order to fully uncover cellular mechanobiological mechanisms and heterogeneity in aging, emerging single-cell and single-molecule technologies are essential for the study of the extent and cell-to-cell variation of age-related deterioration of cellular mechanobiological characteristics, including force, cytoskeleton and nuclear architectures, and mechanotransduction signaling. These novel techniques thus will allow us to probe pico- to nano-newton forces, along with spatiotemporal mechanical analysis and modeling of molecular and cellular mechano-sensing and -transduction in single cells, so as to establish mechanistic frameworks to understand the mechanobiological impacts of aging and develop new strategies to halt and reverse the aging process.