PHYSIOLOGY OF AGEING OF THE MUSCULOSKELETAL SYSTEM

Dr Katalin Boros MD PhD FRCPath

Adult Histopathology, Department of Cellular Pathology

Central Manchester University Hospitals Foundation Trust

Clinical Sciences Building 1

Oxford Road

Manchester

United Kingdom

M13 9WL

Email: katalin.boros@doctors.org.uk

Prof Tony Freemont BSc MD FRCP FRCPath*

MMPathIC, The Manchester MRC/EPSRC Molecular Pathology Node

Faculty of Biology Medicine and Health

University of Manchester

And Manchester NIHR Biomedical Research Centre

Central Manchester University NHS Foundation Trust

Manchester

United Kingdom

M13 9NQ

Email: Anthony.Freemont@cmft.nhs.uk

Tel: 0161 3065326

*Corresponding author

Abstract

This review aims to provide a summary of current concepts of ageing in relation to the

musculoskeletal system, highlighting recent advances in the understanding of the mechanisms

involved in the development of age-related changes in bone, skeletal muscle, chondroid and fibrous

tissues. The key components of the musculoskeletal system and their functions are introduced

together with a general overview of the molecular hallmarks of ageing. A brief description of the

normal architecture of each of these tissue types is followed by a summary of established and

developing concepts of mechanisms contributing to the age-related alterations in each. Extensive

detailed description of these changes is beyond the scope of this review; instead, we aim to highlight

some of the most significant processes and, where possible, the molecular changes underlying these,

and refer the reader to in-depth, subspecialist reviews of the individual components for further

details.

Key words: Connective Tissue; Ageing; Musculoskeletal; Physiology

Total word count: 9,831

A) INTRODUCTION

The musculoskeletal system comprises bone, skeletal muscle, chondroid tissues (articular cartilage,

intervertebral disc, meniscus), fibrous tissues of tendons, ligaments and joint capsule, and fat.

Together, these tissues form the structures that give the organism its shape, enable movement,

protect internal organs, participate in humoral signalling and provide a reserve for organic and

inorganic molecules (e.g. fat, glycogen, amino acids, calcium and phosphate), which are key to

homeostasis. While the functions and morphological features of each of these tissues are clearly

distinct, they share some components and features at the cellular (e.g. common mesenchymal stem

cells as precursors for osteoblasts, chondrocytes and adipocytes) and extracellular (e.g. collagen and

elastin fibres in bone, cartilage and fibrous tissues) level and overlap in the molecular signalling

networks and mechanisms within them. Furthermore, these tissues interact closely and together

form complex anatomical and functional structures (e.g. joints, muscle-bone units) to execute their

functions.

Ageing can be defined as ‘the inevitable time-dependent decline in organ function that eventually

leads to death’ [1]. In general terms, the processes of ageing affect the balance of mechanisms,

which ensure homeostasis within organs, and alter the tissue response to injury. At a molecular

level, ageing is associated with accumulation of genetic damage [2]. Nine hallmarks of ageing have

been proposed, which are linked to each other and can be grouped into three subcategories

(primary, antagonistic and integrative), illustrating their connections and relationship to each other

[3].

In this categorisation, genomic instability, telomere attrition, epigenetic alterations and loss of

proteostasis form the cause of damage (primary hallmarks). They elicit antagonistic or compensatory

mechanisms, which form the second group (antagonistic hallmarks): deregulated nutrient sensing,

mitochondrial dysfunction and cellular senescence. A common feature of antagonistic hallmarks is

that although they are initially beneficial, if present chronically and at elevated levels, they lead to

further damage. Ultimately these changes lead to phenotypic alterations associated with the third

group, or integrative hallmarks: stem cell exhaustion and altered intercellular communication, which

have a direct effect on tissue function and homeostasis.

The complex connections between the individual hallmarks go beyond the above hierarchical model

and are not yet fully understood. These mechanisms represent the current concepts of an evolving

body of knowledge on ageing. They are in themselves not specific to physiological processes, and

many of them also play a role in various diseases.

When considering age-related changes within the musculoskeletal system, it is helpful, from the

clinical point of view, to consider these starting from the aspect of the organism and progress

towards the cellular and molecular changes within the individual components of the system. Thus, it

is well recognised that ageing results in alterations in the shape of the organism (loss of muscle and

bone mass, changes in bone geometry, alterations in the relative proportion and distribution of fat),

changes in its movement (decline in the coordination, speed and strength of movements, and load-

bearing capacity), reduced protection to internal organs (increased risk of bone fracture, decreased

healing capacity), alterations in the components of humoral signalling (changes in the amount of,

response to and metabolism of vitamin D, parathyroid hormone (PTH), inflammatory cytokines and

sex hormones) and decline in reserve/storage of organic and inorganic molecules (reduced fat,

glycogen, protein/amino acid, calcium and phosphate stores). In the following sections, we consider

how these changes relate to the individual components of the musculoskeletal system, specifically

bone, skeletal muscle, cartilage and fibrous tissues. A brief description of the cellular and molecular

mechanisms underlying these phenotypic alterations is included, highlighting recent advances in the

field of musculoskeletal research. An extensive amount of research has been conducted in each of

the areas touched upon in this review, and the reader is referred to in-depth specialist publications

for details.

A) AGEING IN BONE

The morphologic and systemic features of bone ageing are as follows: loss of bone mass and mineral

content, alterations in bone shape and geometry, increase in bone marrow fat content, increased

risk of fractures and reduced healing capacity, altered response to growth factors and hormones,

and reduced calcium and phosphate stores. The following sections outline our current

understanding of how the key constituents of bone are altered during ageing, together leading to

these changes.

B) Adult bone constituents:

A bone is composed of a mineralised organic matrix within which are located multiple cell

populations, including those integral to and directly involved in tissue maintenance and function

(skeletal/mesenchymal stem cells, osteoblasts and their terminally differentiated form, the

osteocytes, and osteoclasts) and those which support the function of, or are precursors for cells of,

the haematopoietic system (bone marrow components – not discussed further here). Osteocytes

constitute 90–95% of the total integral cell population.

The structure and microarchitecture of bone are such that the integral cell populations are relatively

sparsely distributed as clusters of and single cells within the matrix (e.g. osteoblasts, osteocytes),

while others are located together in the same compartment (e.g. haematopoietic cells and

mesenchymal stem cells). The cells communicate with each other through direct cell-cell interaction

(e.g. during mechanotransduction through gap junctions between osteocytes) and/or secreted

factors (e.g. stem cells and progenitors, mesenchymal stem cells and osteoclasts – discussed below).

B) Bone cellular components and ageing

C) Mesenchymal/skeletal stem cells:

Osteoblasts, osteocytes and osteoclasts form the populations of cells directly involved in the

framework and maintenance of mature bone tissue and its functionality as part of the

musculoskeletal system. Osteoblasts and osteocytes are derived from the differentiation of bone

mesenchymal stem cells/skeletal stem cells (BMSC/SSC), while osteoclasts originate from precursors

derived from haematopoietic stem cells (HSC) [4]. Skeletal stem cells are multipotent self-renewing

progenitors that serve as progenitor cells for cartilage, bone, bone marrow stroma and bone marrow

adipocytes. They play a regulatory and controller function within the bone marrow

microenvironment, influencing vascular and haematopoietic functions and osteoclastogenesis [5].

Key transcription factors in the regulation of progenitor lineage commitment include Runt-related

transcription factor 2 (RUNX2 - osteogenic), sex-determining region Y-box 9 (SOX9 - chondrogenic)

and peroxisome proliferator activated receptor γ2 (PPARγ2 - adipogenic). All are regulated by

members of the transforming growth factor β/bone morphogenetic protein (TGFβ/BMP)

superfamily, wingless type MMTV integration site (WNT) and sonic hedgehog (SHH) [5].

SSCs reside in the perivascular regions of the external surfaces of bone marrow sinusoids, where

they interact closely with HSCs [6]. Because of their multiple functions, SSCs play a key role in bone

development, growth, modelling and remodelling. Bone formation takes place through

differentiation of skeletal stem cells through lineage-committed progenitor cells into osteoblasts, a

proportion of which ultimately become osteocytes (discussed in further detail below).

Most of our understanding of age-related changes in SSCs to date has been compiled from in vitro

studies. While in vitro experiments provide useful insight into the mechanisms of SSC function, there

are significant differences in the cellular microenvironment and cell function between in vitro and in

vivo conditions, which must be considered when applying these data to the living organism

(reviewed in Ref. [7]). In vitro cultured SSCs are rapidly expanded through population doubling and

show accelerated ageing during this process, while SSCs in vivo are slow cycling. Furthermore,

differences in the sources of SSCs, methods for their isolation, culture conditions and analysis mean

that there is a lack of conclusive data from in vitro studies. Thus, while self-renewal and

differentiation potential, presence of senescence markers (e.g. β-galactosidase), ageing indices such

as telomere length, reactive oxygen species, superoxide dismutase levels and energy metabolism

have been studied in vitro, the results are often contradicting and the overall trend appears

heterogeneous [7–9]. Nonetheless, it has been established that the number of SSCs decreases from

childhood until the age of 30 and is then stable [10]. Furthermore, there is an increased propensity

towards adipogenic differentiation in MSCs from old donors [11–13]. While the detailed mechanisms

for this ageing-associated differentiation bias are not completely understood, recent studies have

implicated miRNAs to be involved in this process [14].

The stem cell microenvironment plays a crucial role in stem cell function, both as an effector site for

signalling molecules secreted by stem cells, including regeneration-enhancing molecules, growth

factors and inflammatory modulating factors, and as a source of regulatory signals that modify stem

cell functions [15,16]. A key bone regulatory pathway is the canonical WNT pathway, which has an

anabolic effect, in part due to the repression of chondrocyte and adipocyte differentiation pathways

in MSCs, through the promotion of osteoblastogenesis, osteoblast proliferation, differentiation and

mineralisation activity [17]. Ageing is associated with the downregulation of the expression of

various WNT proteins, co-receptors and inhibitors, suggesting an altered ratio of inhibitors to

effector proteins that leads to reduced osteoblastogenesis [18]. These alterations could contribute

to bone loss through reduced osteoblast formation, altered signalling and interaction within the

local niche, and changes in the levels of factors secreted by SSCs, affecting osteoclast differentiation

and activation [4].

C) Osteoblasts and osteocytes

Osteoblasts are osteogenic cells that synthesise bone matrix and enable its mineralisation. They are

recruited to areas of bone resorption (resorption pits), where a small percentage of them ultimately

become surrounded by the mineralised matrix as single cells known as osteocytes. Osteocytes are

connected to each other by gap junctions between the cytoplasmic processes that they extend along

bone canaliculi.

Osteocytes are the mechanosensor cells in bone and respond to shear forces generated by fluid flow

due to mechanical loading, which also has an anti-apoptotic effect on osteocytes. Osteocytes also

produce factors that control bone resorption (such as the cytokine receptor activator of nuclear

factor B ligand (RANKL) [19,20] and influence phosphate homeostasis (via signalling through dentin

matrix acidic phosphoprotein 1 (DMP1) and fibroblast growth factor 23 (FGF23) [21,22]. They also

secrete sclerostin, a WNT-antagonist, thus controlling bone formation [23,24].

Ageing is associated with a reduction in the number of osteoblasts, a reduction in osteoblast

proliferation and increased apoptosis [25,26]. The ability of cells to sense and respond to mechanical

forces also diminishes with age, which, in osteocytes, results in increased apoptosis, susceptibility to

mechanical damage, changes in intracellular signalling and gene expression regulation [27,28].

C) Osteoclasts

Osteoclasts differentiate from myeloid precursors derived from haematopoietic stem cells. Their

function is the resorption of bone, which is controlled by signalling from osteoblasts, osteoclasts and

MSCs/SSCs. Osteoclastogenesis increases with ageing, with expansion of the osteoclast pool and

alterations of the relationship between osteoclasts and osteoblasts [29]. There is thus a relative

increase in osteoclast numbers with ageing compared to osteoblasts, contributing to the increase in

bone resorption. As indicated above, osteocytes regulate osteoclast activity and differentiation, and

the increased presence of dying osteocytes with ageing may also contribute to increased osteoclast

activity. Furthermore, studies have also suggested that age-related alterations in bone matrix may

contribute to altered osteoclast activity with ageing, although studies have shown opposing effects

on osteoclast activity [30,31].

Effect of cellular alterations in ageing bone

A key physiological process influenced by the above-described alterations in SSCs, osteoblasts,

osteocytes and osteoclasts is bone remodelling. The development of microcracks and microfractures

within bone due to loading occurs throughout life, secondary to mechanical stress. Microcracks are

believed to develop because of the disruption of the architecture of the mineral and/or organic

extracellular components, compounded by a disruption of osteocyte architecture [32]. This

microdamage increases exponentially with age [18]. Micro damaged bone is removed and replaced

with new bone through the process of bone remodelling. Thus, throughout adult life, there is a

constant cyclic renewal of bone, which results in the entire skeleton being replaced by new bone

every decade [33]. Bone remodelling is dependent on the balanced activity of bone-forming units

(BFUs), which consist of osteoclasts and osteoblasts recruited to the foci of new bone formation.

Following the initial resorptive activity of osteoclasts, which remove areas of damaged bone, new

bone is synthesised by activated osteoblasts. There are site-specific variations in the rate of bone

turnover, but the overall outcome in healthy adults is the replacement of all the removed bone with

new bone. With ageing, there is an increase in bone turnover and a disruption of the remodelling

activity, predominantly as a result of altered osteoblast recruitment and function [22]. This leads to

decreased bone formation and a net loss of bone.

In parallel with the ongoing repair activity stimulated by microdamage, there are also changes in the

shape and geometry of bone in response to loading, hormonal and growth factor signalling [34–36].

This occurs through periosteal deposition and endosteal resorption of bone in a process referred to

as cortical drift. In cortical drift, endosteal resorption exceeds endosteal bone formation and can

exceed periosteal bone deposition, resulting in cortical thinning. The initial pre-pubertal period of

rapid cortical shift levels off in the adult, then increases again in the elderly. Thus, with ageing, there

is reduction of periosteal bone formation and increase of endosteal resorption, leading to cortical

thinning, increased cortical porosity, trabecular thinning and loss of trabecular connectivity. These

changes ultimately result in the reduction of bone strength [18].

B) Bone extracellular matrix (ECM) and ageing:

Bone matrix is composed of the organic component, consisting predominantly of type 1 collagen and

approximately 5% (weight) non-collagenous proteins, and the mineral component hydroxyapatite.

The mineral component is not pure and contains trace contents of a number of different materials,

depending on the organism's nutrition. The triple-helical collagen fibrils secreted by the cells form

larger fibrils and fibres; these are stabilised by cross-links, which form within and between the fibrils.

Cross-links can be formed enzymatically (lysyl hydroxylase and lysyloxidase) – these are present

between the C- or N-terminus of one collagen molecule and the helical region of the other.

Enzymatically formed cross-links mature during development, and from reducible divalent cross-

links, they are converted to non-reducible trivalent cross-links, resulting in increased collagen

stiffness [18]. This process is limited to the period of growth and maturation of the organism and

does not change with ageing. However, non-enzymatic cross-links also occur and include the

advanced glycation end products (AGEs), which are induced by glycation or oxidation. Studies have

indicated that AGEs are more numerous with age than enzymatic cross-links [37–42].

Collagen cross-linking is believed to play a role in the mechanical properties of bone and is also

associated with the rate of mineralisation [43]. Cross-link formation also affects the way

microdamage is propagated. Furthermore, removal of AGEs is believed to be possible only by bone

resorption, and their presence increases osteoclast activity while decreasing the activity of

osteoblasts, thus contributing to bone fragility [18,38].

Compounding these effects, ageing is also associated with decreased total protein production in

bone [44], with an increase in protein fragmentation in older individuals. There are age-dependent

changes in the expression and distribution of non-collagenous proteins and in the extent of their

post-translational modifications. Post-translational modifications are key to the normal functioning

of these proteins, and the changes lead to a decline in their multifunctionality, further altering the

extracellular microenvironment [45,46].

A frequently used measurement for bone mineral content is bone mineral density (BMD), which has

been shown to decline with advancing age in both men and women, with men showing a gradual

decrease throughout adult life and women showing acceleration of decline for a few years following

the menopause, after which the rate of decline stabilises [47]. Bone mineral crystals are nano-

crystals that contain inclusions and substitutions, and these also show variation with age. X-ray

diffraction analysis of homogenised biopsies from the iliac crest of patients between a range of 0–95

years has shown that the mineral undergoes maturation with age, with increase in crystal size and

perfection until age 25–30 years, followed by decrease and slight increase again in the oldest age

group [48,49]. However, this study did not distinguish between locations within bone or sex

differences, which are also known to be associated with variation in BMD [50].

B) Humoral factors contributing to age-related changes in bone

one formation and remodelling are influenced by several humoral factors, and bone cells

communicate and receive signals through extensive autocrine, paracrine and endocrine networks.

We highlight three key networks below: the growth hormone-insulin-like growth factor (GH-IGF)

signalling axis, sex hormones and inflammatory cytokines.

C) Growth hormone – insulin-like growth factors (GH-IGF) axis

IGFs are mediators of the effect of GH and play a role in regulation of growth, development and

lifespan. Mouse models of IGF1 knockout in osteoblasts, osteocytes and osteoclasts have been

developed. Conditional knockout of IGF1 in osteoblasts during development leads to decreased bone

formation and reduction in bone mass, and these studies highlight the requirement for IGF-1 for PTH

effect on bone. Data from IGF-1 studies in osteoblasts show that this factor is required for SSC

proliferation and differentiation to osteoblasts and for the biosynthetic activity of osteoblasts. IGF-1

knockout in osteocytes also affects bone growth. In addition, IGF-1 is required for osteoclast

differentiation – this is suggested by the finding that global IGF-1 knockout mice show increased

bone volume to total volume ratio due primarily to a reduction in osteoclast numbers [51]. Ageing is

associated with a gradual decline in secreted GH and an accompanying fall in the levels of circulating

IGF, along with a concomitant increase in circulating IGF-binding proteins, which sequester IGF and

have direct anti-IGF effects. Furthermore, aged cells appear to have reduced sensitivity to IGF-1

signalling [52]. Thus, reduced signalling through the GH-IGF axis associated with ageing contributes

to reduction in bone mass.

C) Sex hormones

In women, the postmenopausal period of rapid loss of bone mass is attributed to the decrease in

oestrogen levels. Oestrogen has an inhibitory effect on osteoclasts, and the release of this inhibition

leads to increased bone turnover. Increase in osteoclast activity elicits an increase in osteoblasts, but

this is insufficient, and the net effect is excessive bone resorption, with loss of bone in both the

cortical and trabecular compartments [52]. Because oestrogen is derived from aromatisation of

testosterone, decreased levels of androgens in men with ageing also contribute to bone loss, in part

due to the above mechanism. In keeping with this, the free serum levels of oestradiol correlate

better with BMD than testosterone levels in men [53]. In addition, osteocytes and osteoblasts show

decreased oestrogen receptor synthesis with ageing, further compounding the effect of decreased

oestrogen signalling.

C) Cytokines

A number of cytokines act to modulate osteoclast and osteoblast activity. Interleukin (IL) 1α, IL1β,

IL6, IL7 and tumour necrosis factor α (TNFα) have been shown to promote osteoclastogenesis, while

interferon (IFN) β, IFN γ, IL4, IL10, IL13 and IL12 have a negative effect on osteoclast function.

Tumour growth factor β (TGFβ) has both an inducer and suppressor effect on osteoclasts.

Osteoblasts secrete IL6, and TGFβ also has a dual (activator and suppressor) function on osteoblasts.

The observation that ageing is associated with an increase in inflammatory cytokines has led to the

development of the concept of ‘inflamm-ageing’, i.e. the development of a chronic inflammatory

state with age, triggered by a lifetime exposure to antigens and leading to release of inflammatory

cytokines, tissue damage and reactive oxygen species. These effects are believed to contribute to the

altered homeostatic processes in various tissues of the musculoskeletal system, including bone [54].

The recognition of the interactions between bone and the immune system has led to the

development of the field of osteoimmunology [55]. Because inflammatory processes and elevated

levels of cytokines have been found to play a role in degenerative disorders, elucidation of the

mechanisms of inflamm-ageing contributes to our understanding of the development of age-related

diseases and may identify potential targets for therapy.

A) AGEING IN MUSCLE

Ageing is associated with a progressive loss of skeletal muscle mass and function, which together

form the syndrome of sarcopenia. Sarcopenia is associated with a risk of adverse outcomes including

inferior quality of life, physical disability and death. The European Working Group on Sarcopenia in

Older People recognises multiple causes of sarcopenia, with primary sarcopenia defined as that

which has no other underlying cause apart from ageing [56]. Sarcopenia overlaps with the geriatric

syndrome of frailty and is a cause of weight loss, impaired movement and weakness in the elderly

[57]. The following section outlines the key changes associated with ageing of skeletal muscle and

their impact.

B) Components of skeletal muscle and ageing

Skeletal muscle is composed of myofibres, which are multinucleated syncytial cells that contain

contractile proteins in their cytoplasm. There are two main types of myofibres, classified as slow

twitch (type I) and fast-twitch (type II), according to whether they use aerobic (type I) or anaerobic

(type II) metabolism. The muscle-committed progenitors, which are responsible for the repair and

growth of myofibres, are known as satellite cells and are located beneath the basal lamina of mature

myofibres. In addition to the cellular components, a key element of muscle function is its

innervation, which is realised at the neuromuscular junction.

C) Loss of muscle mass

The loss of muscle mass associated with ageing is due to atrophy of muscle fibres. The rate of muscle

mass loss is 3–8%/decade from age 30 and increases further after age 65 [58]. This atrophy is largely

due to loss of myofibrillar protein, which is more pronounced in fast (type II) and less so in slow

(type I) fibres [59]. There is reduced synthesis of myofibrillar and mitochondrial proteins with age

[60]. This is in part due to the endocrine changes associated with ageing, namely reduced production

of anabolic cytokines including IGF1 in ageing muscle [61]. Aged muscles also show ‘anabolic

resistance’, which means that anabolic stimuli such as exercise or ingestion of amino acids, which

normally induce protein synthesis, elicit a reduced response [62]. Interestingly, catabolic stimuli such

as glucocorticoid hormones also have a reduced effect on aged muscle [63]. It is unclear which

cellular mechanisms are responsible for these altered responses.

In addition to atrophy of individual fibres, there is also a general loss of the number of muscle fibres,

which accelerates from approximately 5% between age 24 and 50 years up to 35% over the

following 25 years [64]. One possible mechanism for loss of muscle mass is due to changes in the

neuromuscular junction with age. Denervation of muscle fibres contributes to loss of muscle mass.

Insufficient re-innervation leads to atrophy or apoptosis of muscle fibres [59,65].

Another component of muscle mass loss lies in the function of satellite cells. Satellite cells are

involved in the repair of damaged muscle and possibly play a role in the maintenance of muscle

mass [66]. Satellite cell numbers are reduced with age by up to 50%, leading to a decreased

regenerative capacity of muscle [59,67]. In vitro studies have shown that aged satellite cells show

reduced activation, proliferation, colony formation and differentiation [68]. Aged satellite cells are

also more prone to undergo senescence and apoptosis [69]. Furthermore, higher levels of fibroblast

growth factor 2 (FGF2) have been demonstrated in ageing. FGF2 is produced by the satellite cell

niche, and an increase in its levels is expected to lead to loss of quiescence and self-renewal ability

of satellite cells, which in turn would also make them more vulnerable to environmental stresses

such as oxidative stress [70]. Similar to the differentiation shift seen in SSCs described in bone above,

aged satellite cells also show increased entry into alternative differentiation pathways, resulting in

fibroblastic and adipogenic differentiation [68]. In addition, increased numbers of fibro-adipogenic

progenitors are also seen in aged muscle [71]. These observations help explain the changes in

muscle composition seen with ageing, namely the presence of increased amounts of fat and

connective tissue.

C) Changes in muscle function

While loss of muscle mass is significant during ageing, loss of muscle strength and function occur to

an even greater extent and have a significant impact. Multiple mechanisms underlie the loss of

function; a key one is the selective loss of fast muscle fibres. This is due to selective loss of fast

motor neurons with ageing. As muscle fibre type is determined by the type of motor neuron

innervating it, loss of fast motor neurons leads to ‘orphan’ fast muscle fibres. These are then re-

innervated by neurons from neighbouring units, leading to their regrouping and partial conversion to

slow fibres, producing a hybrid fibre phenotype or fibre-type switch [64,65,72]. This hybrid

phenotype causes a disruption in the normal recruitment of motor unit and loss of the normal

intermixed pattern of muscle fibre types, leading to a deterioration of motor skills. In addition, the

increase in fibrofatty tissue within skeletal muscle with age described above also contributes to the

disruption and disorganisation of its architecture and subsequent reduction in function.

Ageing is also associated with intrinsic changes in muscle fibres. These include changes and defects

in mitochondrial function, including increased generation of reactive oxygen species [73] and

changes in the function and relative amounts of mitochondrial proteins. These and other changes

(summarised in a review by Demontis et al.) are considered to be the factors leading to lower

respiratory capacity, decreased ATP levels, decreased fatty acid metabolism, intracellular

accumulation of lipids and eventual insulin resistance seen in the aged [59]. These results were,

however, obtained by the study of purified mitochondria; data obtained from studies using

permeabilised fibres is not conclusive, possibly due to differences in experimental settings. Further

age-related metabolic defects intrinsic to myofibres include increased glycolysis, decreased glucose

uptake and decreased glycogen synthesis [74].

Ultrastructural and molecular changes observed in aged muscles indicate that there is impairment of

calcium storage and release and a decline in calcium homeostasis, which in turn affects muscle

function. Contraction causes mechanical stress and potential destabilisation of the muscle

membrane. Hypomorphic mutations of dystrophin and dysferlin genes, which act as part of the

network that ensures membrane repair and stability, are believed to affect plasma membrane

stability in old age [75].

As mentioned before, there is a decrease in the synthesis of myofibrillar proteins with ageing. In

addition, these proteins are also affected by functional changes including reduced ATPase activity of

actomyosin and alterations of myosin isoform expression. A knock-on effect of these changes is that

they would ultimately affect the distribution and function of sarcomere-associated signalling factors,

which may result in an even further-reaching effect on muscle function due to altered intercellular

communication [59].

A phenomenon associated with ageing is the induction of various pathways of the stress response

mechanism – these are activated in response to protein and DNA damage occurring with time.

Increased expression of the chaperone-dependent ubiquitin ligase CHIP has been shown in

sarcopenic rats [76]. This protein catalyses the degradation of misfolded proteins, and studies with

CHIP knockout mouse models have shown accelerated muscle mass loss during ageing [77]. While

activation of stress response pathways is initially a protective and beneficial mechanism, excessive

activity may have detrimental effects and could ultimately lead to degradation of not only

misfolded/non-functional proteins but also functional myofibrillar proteins, further compounding

the protein loss seen with ageing. An example of this is seen in the effects of the Forkhead box

protein O (FOXO) transcription factors, which are central regulators of the stress response pathways

and, in muscle, are important factors in protein homeostasis. Varying levels of FOXO activity appear

to have distinct and opposing outcomes in terms of age-related changes in protein homeostasis

[59,78].

C) The muscle-bone relationship and ageing

Muscle and bone form a unit for movement, and muscles are functionally matched to the bones

they move in size and geometry. As the capacity of muscle to generate force declines and the

anabolic response of bone to muscle-derived stimuli changes, this muscle-bone relationship is

altered. Data show that exercise can enhance bone mass and strength in young animals but not in

older ones. The reduced osteocyte number and density seen with ageing, which leads to impairment

of the signalling network, is considered to be the likely cause for this alteration in response [79].

Skeletal muscles secrete myokines, signalling molecules that have a positive effect on bone

formation and an inhibitory effect on osteoclast differentiation [80]. Muscle contraction is a

stimulant of myokine secretion. The reduction of muscle protein synthesis and overall muscle

function seen in ageing may affect myokine synthesis and secretion. The profile of secreted

myokines is also hypothesised to change with ageing. Thus, alterations in paracrine signalling are

also believed to contribute to changes in the musculoskeletal unit with ageing [79].

A) AGEING IN CHONDROID TISSUES

Two types of chondroid tissue are widely studied with respect to age-related changes: articular

cartilage and intervertebral disc. The structure and composition of these differ, with respect both to

their extracellular matrix (ECM) and cellular components. The two structures will therefore be

discussed separately in the following sections.

B) Articular cartilage

Articular cartilage is unique in its architecture in that the only cell population is the chondrocyte

population, with no innervation, vascularisation or macrophages present within the tissue. The

chondrocytes are embedded in an ECM composed of proteoglycans, type II collagen, lubricin and

hyaluronic acid [81]. Articular cartilage chondrocytes have a very low rate of replication and are

therefore highly vulnerable to the ageing process.

Articular cartilage can be divided into 3 zones, beginning with the superficial zone (at the joint

surface), followed by the mid- and the deep zones, under which lies the subchondral bone. Each

zone differs in its chondrocyte population and matrix composition. A difficulty in the study of ageing

cartilage has been the separation of age-related changes from pathological conditions in which

ageing is a risk factor, such as osteoarthritis. Studies of cartilage damaged by osteoarthritis have

reported the presence of stem/progenitor cells in isolates from animal and human superficial zone

cartilage [82,83]. Chondroprogenitors may be responsible for the repair of damaged cartilage by

migration to areas of damage. Apart from the superficial cartilage zone, chondroprogenitors may be

found in synovial tissue, bone marrow and the articular fat pad in osteoarthritis [84,85]. However, no

direct evidence that these progenitors support repair has yet been obtained.

The cell density of cartilage decreases with ageing – progressive involution of chondrocytes from age

40 onwards has been shown by histochemical, histometrical and electron microscopy studies.

Chondrocyte involution mainly affects the superficial zone and is associated with a reduction in the

numbers of lacunae containing 3–4 cells and an increase in the numbers of lacunae containing single

or no cells. Between age 40 and 80 years, approximately 50% of chondrocytes are lost [81,86].

Apoptosis has been suggested to account for the decrease in chondrocytes in ageing (and in

osteoarthritis) [81]. The transcriptional regulator non-histone protein high mobility group (HMGB2)

shows specific expression in superficial zone cartilage and is associated with chondrocyte survival.

Ageing entails a reduced and eventual loss of expression of HMGB2, which in turn has been shown

to increase chondrocyte susceptibility to apoptosis [87,88]. However, it is not entirely clear to what

degree apoptosis contributes to the reduction of chondrocyte numbers with age and what is the role

of other possible signalling mechanisms such as mitochondrial dysfunction and ROS production,

hypertrophic or terminal differentiation, or autophagy are [81]. An alternative explanation to

reduced function is offered by the theory that it is metabolic failure in living cells, rather than

increased cell loss, that leads to these changes. Evidence supporting this includes the finding that

aged chondrocytes exhibit altered or blunted responses to growth factors and stressors, including

reactive oxygen species. Changes in anabolic and proliferative responses to TGFβ, basic fibroblast

growth factor (bFGF), platelet-derived growth factor (PDGF) and IGF-1 have also been demonstrated

[52]. Oxidative stress has also been postulated to be the cause of telomere shortening and other

senescence-associated markers found in ageing chondrocytes. Changes or defects in autophagy have

been implicated as a mechanism involved in age-related changes in articular cartilage [81,89].

The ECM of articular cartilage is composed of proteoglycans, hydrophilic macromolecules that

imbibe water. The water content associated with proteoglycans accounts for the internal swelling

pressure of cartilage and contributes to its function as a shock absorber/spacer within the joint. The

ECM changes in amount and composition with age, with reduction of water content and

fragmentation of the protein components of proteoglycans and other glucosaminoglycans (GAGs). In

addition, there is increased fragmentation of collagen with age, which leads to reduced tensile

strength. The quality of the ECM constituents changes as well, with alteration in the sulphation

patterns of GAGs, shifts in the ratios of collagen types present and changes in the crosslinks formed

between collagen molecules. AGEs form within cartilage, and other age-related alterations in the

ECM are also seen (reviewed in Ref. [52]).

B) Intervertebral disc

Intervertebral discs (IDs) serve as a spacer and shock absorber between vertebrae, providing

flexibility and support to the spinal column [90]. IDs are composed of the central nucleus pulposus

(NP), surrounded by the annulus fibrosus (AF). Age-related changes firstly and primarily affect the

NP, which consists predominantly of ECM composed of hydrated proteoglycans surrounded by a

loose, irregular network of type II collagen fibres and elastin. Cells constitute only 1% of the NP and

are believed to comprise two main populations with different ontogenic origins – one derived from

notochordal cells and the other from cells that migrate into the NP [52,90]. Studies have shown a

molecular cross-talk between these two cell populations, which enables homeostasis and

maturation of the NP, cell survival and ECM synthesis [52]. Notochordal-derived cells decrease in

number with ageing, and the loss of their protective and anabolic role is therefore believed to

contribute as one of the initial events in degenerative changes in the NP. In association with the loss

of notochordal-derived cells, there is activation of ageing-related cellular responses such as

apoptosis, senescence and autophagy. These are protective mechanisms, the activation of which

protects NP cells from cell death by allowing for compensation for nutrient deficiency (autophagy)

and withdrawal from the cell cycle (senescence) and thus evasion of programmed cell death [52].

The cellular changes occur in parallel with, and are linked to, changes in the ECM. TGFβ and

connective tissue growth factor/CCN family member 2 (CTGF/CCN2) accumulation leads to activation

of a fibrotic repair response in NP cells and subsequent deposition of type I collagen fibres replacing

the normally found type II collagen there. Proteoglycan synthesis also changes, with a shift from

aggrecan to versican, biglycan and decorin. There is also increased expression of matrix

metalloproteinases and proteins belonging to the A Disintegrin and Metalloproteinase with

Thrombospondin Motifs (ADAMTS) family, which leads to degradation of proteoglycans and

ultimately alteration of the biochemical properties of the ECM. This is paralleled by increased

expression of factors that promote ingrowth of vessels and neurites, further altering the

composition and biomechanical properties of the disc [91]. These processes lead to reduced osmotic

pressure and water content, with subsequent loss of the ability to evenly transfer mechanical forces.

The resulting uneven forces on the AF lead to microtrauma and tears, and the NP may eventually

herniate through the fibrous ring.

Recent studies have highlighted a role of circadian clock mechanisms in the degeneration processes

of ageing intervertebral discs [92]. IVDs exhibit a diurnal cycle of alternating higher loading (activity

phase) and low load recovery (resting phase). The alternation of these cycles causes fluid flow from

the NP through the AF and into the vertebral cartilaginous end plate (CEP) and vice versa, enabling

exchange of nutrients and metabolites, which is crucial for disc homeostasis. Using mouse and

human NP cell cultures and a tissue-specific knockout mouse model of an essential clock component,

Brain and Muscle Arnt-like protein 1 (Bmal1), Dudek et al. established the existence of autonomous

circadian clocks in mouse and human IVD cells. The activity of these cells dampened with age and

was dysregulated by catabolic cytokines. Genetic disruption of the clock predisposed the animals to

IVD degeneration. Analysis of the circadian transcriptome of the IVD tissue indicates that circadian

rhythm is a critical regulatory mechanism for IVD biology. Proinflammatory cytokines disrupt the

circadian clock, and this disruption may be a driver of the catabolic response in proinflammatory

states. This study looked at the effect of the proinflammatory cytokine IL-1β and found that it

disrupted the circadian clock mechanism at the single cell level [92]. In a separate study, Le Maitre et

al. established that both IL-1α and IL-1β are synthesised by native disc cells in non-degenerate and

degenerate IVDs; its receptor, IL-1R, is expressed by NP cells in both non-degenerate and degenerate

discs, with an increase in IL-1R expression in degenerate tissue [93]. IL-1 is implicated in the switch in

collagen and the altered proteoglycan synthesis seen in degenerate discs and was also shown to

possibly play a role in chondrocyte phenotype change from chondrogenic to fibroblastic.

Furthermore, in degenerate discs, IL-1 was found to have a positive feedback effect on its expression

in IVD cells, while in non-degenerate cells, it showed negative feedback. These findings parallel those

seen in articular cartilage in osteoarthritis.

A) FIBROUS TISSUES AND AGEING

Tendons, ligaments and joint capsule form the main fibrous tissue components of the

musculoskeletal system. All of these are highly organised structures that contain fibroblasts, collagen

(predominantly type 1, with a lesser number of minor collagens), proteoglycans, glycosaminoglycans

and elastin. With age, these tissues undergo degenerative changes, which increase susceptibility to

partial or complete disruption. In addition, because of their key role in joint movement, these

structures also participate in proprioception.

B) Tendons and ageing

Tendons link muscles and bones together and serve to transmit loads and/or motion, thus

participating in the stabilisation and movement of joints. Tendons receive vascular supply from the

musculotendinous junction and osseotendinous junction, as well as surrounding tissues. Changes in

tendons in ageing include decreased blood flow and loss of normal homeostatic mechanisms due to

increased/repetitive mechanical load, which leads to increased degradative enzyme production and

apoptosis. Under stimulation following injury due to overloading can also compound the

degenerative changes as isolated tendon fibrillar damage leads to changes in the cell-matrix

interactions, which results in the mechanobiological understimulation of tendon cells, leading to

upregulation of collagenase mRNA expression and protein synthesis [94]. As for the other previously

described tissues, there are alterations in tendon ECM as well, with reduction of the ECM volume

(and concurrent increase in relative number of cells per unit tissue), disorientation of collagen fibres,

and increased variation in fibre thickness associated with increased collagen, reduced

mucopolysaccharide and decreased water content. Tenocyte morphology and function also changes,

with tenocytes becoming longer and thinner and exhibiting a reduction in their protein synthesis.

Progenitor stem cells decrease in number (by up to 70%), show lower levels of cell proliferation and

delayed cell cycle progression in older tendons. There is decreased cell growth and stem cell

potential. These alterations lead to impaired healing response. Histopathological changes associated

with tendon degeneration were found in 40–50% of patients over age 40 in a study looking at

rotator cuff and lateral epicondyle tendons from cadavers [95]. However, it is not clear whether

normal ageing is always synonymous with changes in biomechanical properties, and the current

literature shows differing results.

B) Ligaments and ageing

Ligaments serve to stabilise, restrict and guide joint motions by connecting bone to bone. Ligaments

differ in their location relative to the joint (intra- or extra-articular). The structure of ligaments is

similar, but they show differences in cellular properties, e.g. stem cells in the anterior cruciate

ligament (ACL) are different to those in the medial collateral ligament (MCL) [96]. As described for

ageing tendons, ligaments show increased loss of collagen orientation, changes in ECM composition

and cellularity, decreased metabolism and increased apoptosis. There is also decrease in the

numbers of and changes in the morphology of mechanoreceptors, leading to deficits in

proprioception.

B) Capsules and ageing

Joint capsules are fibrous structures that are attached to the bone around the joint and seal the joint

space, provide passive and active stabilisation, and limit movement of the joint. Capsules often

incorporate tendons and ligaments. At their attachment to the bone, capsules can contain

fibrocartilage, composed of type II collagen and GAGs. Relatively few studies have examined the

mechanisms of age-related change in joint capsules. Morphologically, there is reduced cellularity,

hyalinisation of the area near the articular cartilage, cartilaginous metaplasia and focal myxoid

degenerative change, along with increased villous architecture of the synovial layer [97]. These

changes increase incrementally with ageing, and associated loss of vascularity and areas of

calcification develop. These alterations are associated with extension of the type II collagen present

at the attachment zone into the tendon or ligament [98]. The exact mechanisms of these changes

are not yet clear.

A) Summary

The effects of ageing on the musculoskeletal system can be observed in all its components and lead

to loss of bone and muscle mass, impairment of function, increased risk of bone fracture and

fissuring of fibrous and cartilaginous tissues, impaired healing and regeneration, and reduction in

musculoskeletal coordination and proprioception along with alterations in metabolic and humoral

functions associated with these tissues. For the purposes of this review, we have highlighted some of

the key mechanisms involved in these physiological changes in relation to each tissue type; however,

the components of the musculoskeletal system do not function in isolation and form functional units

comprising, for example, the muscle-bone unit or the multiple components of various joint types. At

a molecular level, our knowledge of the mechanisms involved can be mapped to the proposed nine

hallmarks of ageing; the existing knowledge of the extent and details of involvement by each of

these hallmarks is variable for the different tissue types. Challenges for the study of the ageing

process have included a lack of definition of stem and progenitor cell markers in various tissue types,

challenges associated with the extrapolation of data from in vitro studies to the in vivo setting, lack

of clear separation of physiological and degenerative changes in some tissues, and difficult access to

certain cell populations (i.e. tracing of notochordal cells in IVDs, osteocytes embedded in a

mineralised matrix in bone). Nonetheless, a significant amount of data has accumulated and

continues to be generated in this field. Ageing is associated with a number of debilitating

pathological conditions affecting the musculoskeletal system, including osteoporosis, osteoarthritis

and frailty. Understanding of the physiological processes involved in ageing is therefore an important

contributor to elucidating the mechanisms behind these pathological states and can aid in the

identification and development of possible preventative and therapeutic options.

Practice points

Aging changes both the cellular and extracellular components of the musculoskeletal system

It is important when assessing histological sections of connective tissues to be aware that

“normal” features change with age

Ageing may mimic pathological processes

Understanding age-related changes may reveal potential therapeutic targets for age-related

diseases

Research agenda

The phenotype of connective tissue cells during development and ageing have yet to be fully

defined

There is a pressing need for markers of different sub-populations of stem cells

There is a need to test in vivo in Man concepts derived from in vitro and animal studies

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Conflict of interest statement:

There are no potential conflicts of interest that could inappropriately influence the content of this

article.