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: [email protected]
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: [email protected]
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.