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Scand J Med Sci Sports 2009: 19: 500510 & 2009 John Wiley & Sons A/S

doi: 10.1111/j.1600-0838.2009.00986.x

From mechanical loading to collagen synthesis, structural changes


and function in human tendon
M. Kjr, H. Langberg, K. Heinemeier, M. L. Bayer, M. Hansen, L. Holm, S. Doessing, M. Kongsgaard, M. R.
Krogsgaard, S. P. Magnusson
Department of Orthopaedics, Institute of Sports Medicine, Bispebjerg Hospital and Centre for Healthy Ageing, Faculty of Health
Sciences, University of Copenhagen, Copenhagen, Denmark
Corresponding author: Michael Kjr, MD, PhD, Department of Orthopaedics, Institute of Sports Medicine, Bispebjerg
Hospital and Centre for Healthy Ageing, Faculty of Health Sciences, University of Copenhagen, DK-2400 Copenhagen NV,
Denmark. Tel: 145 3531 6089, Fax: 145 3531 2733, E-mail: m.kjaer@m.ku.dk
Accepted for publication 22 May 2009

The adaptive response of connective tissue to loading in tendon includes the isoform that has so far been thought
requires increased synthesis and turnover of matrix proteins, only to exist in skeletal muscle (mechano growth factor).
with special emphasis on collagen. Collagen formation and The increase in IGF-I and procollagen expression showed a
degradation in the tendon increases with both acute and similar response whether the tendon was stimulated by
chronic loading, and data suggest that a gender dierence concentric, isometric or eccentric muscle contraction, sug-
exists, in that females respond less than males with regard to gesting that strain rather that stress/torque determines the
an increase in collagen formation after exercise. It is collagen-synthesis stimulating response seen with exercise.
suggested that estrogen may contribute toward a diminished The adaptation time to chronic loading is longer in tendon
collagen synthesis response in females. Conversely, the tissue compared with contractile elements of skeletal muscle
stimulation of collagen synthesis by other growth factors or the heart, and only with very prolonged loading are
can be shown in both animal and human models where signicant changes in gross dimensions of the tendon
insulin-like growth factor 1 (IGF-I) and transforming observed, suggesting that habitual loading is associated
growth factor-b-1 (TGF-b-1) expression increases to ac- with a robust change in the size and mechanical properties
company or precede an increase in procollagen expression of human tendons. An intimate interplay between mechan-
and collagen synthesis. In humans, it can be demonstrated ical signalling and biochemical changes in the matrix is
that an increase in the interstitial concentration of TGF-b, needed in tendon, such that chemical changes can be
PGE2, IGF-I plus its binding proteins and interleukin-6 converted into adaptations in the morphology, structure
takes place after exercise. The increase in IGF-I expression and material properties.

Mechanical loading of tendon tissue has been the be  15% and that in elite jumping athletes (volley-
focus of biomechanical interest for several years ball players) as high as 50% (Ferretti et al., 1983;
particularly its force-transmitting and energy-storing Ferretti, 1986; Lian et al., 1996). Recent data in
capacity. More lately, however, the biochemical and humans suggest that tendon tissue is metabolically
molecular adaptive responses of the tendon to load- responsive to tensile loading (Kalliokoski et al. 2005;
ing have drawn more attention, and have challenged Bojsen-Moller et al., 2006).
the traditional view of the relatively inert nature
of the tendon. Thus, studies on the inuence of a
mechanical load on tendon tissue adaptation will be Collagen as a main player in mechanically loaded
important for an understanding of the structural and tendon
functional changes that can occur with changes in
tendon loading, and ultimately for an understanding Tendon tissue is subjected to tensile loading and
of tendon overuse and injury healing. Human move- consists predominantly of brillar collagen molecules
ment comes about from the force created by con- primarily type I and III collagen. Although other
tracting muscles, which is transmitted to bone via matrix proteins are also important components of
aponeurosis and tendon. The human tendon is tendon, it seems logical to focus rst on collagen tissue
loaded with tremendous forces, and is therefore often adaptation with loading and to evaluate whether
subjected to overload injuries, such as patellar or increased loading will lead to increased collagen pro-
Achilles tendinopathy. The prevalence of patellar tein synthesis (Kjr, 2004). Collagen is produced in
tendinopathy among athletes has been estimated to tendon broblasts, which are arranged in parallel

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Tendon adaptation to exercise
along the main direction of tension; the cells have an tion and collagen bril diameter are determined in
elongated shape with attened and elongated nuclei both young and elderly patients and healthy subjects
and long, actin-based cytoplasmic protrusions. The (Miller et al., 2006a, b). The use of stable isotope
tendon broblasts interact with the extracellular ma- techniques to study the incorporation of labelled
trix (ECM) through cellmatrix coupling and form a amino acids into tissue in order to study the kinetics
cellular network throughout the tendon. Theoretically, of collagen has been attempted in tendon, ligament
collagen synthesis can be studied in several ways. and bone (Babraj et al., 2005). Briey, the principle of
Animal studies have used approaches with indirect the direct incorporation technique using the precursor-
determination of collagen synthesis by measuring the product approach applicable on tendon tissue is to
enzymes involved in the formation and processing of label the amino acid, proline, e.g. L-13C-proline or
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collagen (e.g. prolyl-4-hydroxylase), and from such L- N-proline. Proline is abundant in collagen, and is
studies, it has been clearly demonstrated that increased incorporated directly into new collagen proteins.
and decreased loading could increase and reduce the Newly synthesized procollagens are posttranslationally
collagen synthesis, respectively (Kovanen, 1989). hydroxylated at the proline residues (forming hydro-
Using the microdialysis technique, catheters are placed xyproline) before being assembled into the triplehelical
in the region of interest (e.g. around or through a structure. Thus, the collagen-specic hydroxyproline
tendon) and the interstitial concentrations of the will be labelled. Measurement of the enrichment of
procollagen propeptides that are cleaved o in the hydroxyproline from a tendon sample, provides a very
maturation from procollagen to collagen (PICP or specic synthesis measure of collagen protein in the
PINP) of both previously loaded and unloaded ten- tendon tissue (Babraj et al., 2005). Using this method,
dons are determined (Langberg et al., 1999, 2001). This acute exercise has been shown to increase the frac-
technique provides the possibility of determining local tional synthesis rate of collagen in the patellar tendon
changes of PICP or PINP in proximity to a tendon from approximately 0.05%/h to around 0.10%/h
that would otherwise only contribute marginally to within 24 h after exercise, showing a signicant in-
any change in these parameters globally (i.e. changes in crease already after 6 h post-exercise (Miller et al.,
the concentrations of PINP or PICP in the circulating 2005). This corresponds to a collagen synthesis that,
blood stream) (Langberg et al., 2001). More recently, on a 24-h level, increases from around 1% at rest to 2
the use of non-radioactive stable isotopes also pro- 3% after exercise. The collagen synthesis rate remains
vided advantages for a more direct determination of elevated for at least 23 days after acute exercise. An
collagen synthesis in human tissue. Given that repre- interesting nding is that the magnitude of increase in
sentative samples of the tissue (like tendon) can be collagen synthesis with acute mechanical loading
obtained, the direct protein synthesis can be deter- seems to be quite uniform and relatively independent
mined as incorporation of the tracer into the connec- of the magnitude of exercise. As illustrated in Fig. 1, a
tive tissue (Babraj et al., 2005; Miller et al., 2006a, ). doubling of the collagen synthesis occurs in human
Tendon tissue sampling can be performed in humans tendon (patellar and Achilles) 24 h after exercise irre-
by percutaneous tendon biopsies, and has been used in spective of whether heavy strength training, enduring
protocols where protein turnover, mRNA transcrip- leg kicking activity or prolonged running was under-

Tendoncollagen synthesis Procollagen expression


(%/hour) (mRNA/mg tendon) Fig. 1. Collagen synthesis in human tendon
Fold increase Fold increase determined either by microdialysis determined
interstitial procollagen propetide concentra-
4
6 tions or by stable isotope injected tendon
incorporation of labeled proline measured in
the resting state or 24 h after an acute bout of
4
exercise. The changes are expressed as fold
2 increase and exercise is either 36 km of running
in athletes (Runn), 1 h of knee extension ex-
2
ercise against resistance to fatigue (Knee) or
three series of eight repetitions of leg press/knee
extension at a load of 70% of one repetition
maximum (Strength). In addition, data for
mRNA of procollagen are provided from ani-
mal experiments where limb muscle was either
subjected to concentric, isometric or eccentric
electrically induced exercise. Data are taken
Rest Runn. Runn. Knee Strength Rest Conc. Ecc. Isom. from Langberg et al. (1999), Heinemeier et al.
36 km 36 km 60 min 8x70% RM 1100 2000 1600 (2007a, b), Miller et al. (2005) and H. Langberg
Achilles Patella Patella Patella g/sec g/sec g/sec and L. Holm, unpublished observation.

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Kjr et al.
taken. However, the present data do not reveal the the tendon. This was visualized by Ralphs et al.
lower limit that exists for stimulation of tendon (2002) in chicken tendon, and also observed in our
collagen synthesis; they suggest that mechanical load- laboratory in human samples (M. Bayer, unpub-
ing sets collagen synthesis at a new level, suggesting an lished observation). Notably, the organized arrange-
on-o switch for connective tissue with regard to the ment of actin laments in human tendon is severely
need for mechanical loading in order to synthesize new disturbed in injured tendon samples. To study the
collagen optimally. Both a single loading bout as well actin cytoskeleton and associated molecules in vitro,
as long-term habitual loading produce a markedly tendon broblasts in culture have been examined and
elevated collagen synthesis response (Langberg et al., shown to form a dense actin cytoskeleton with
1999, 2001; Miller et al., 2005). However, to what laments arranged in parallel. This structure under-
extent this elevated synthesis yields incorporation of goes rapid re-structuring following interference of
collagen into the load-bearing structure of tendon, and cellular integrity, e.g. through experimentally indu-
therefore either an increase in tendon size (hypertro- cing cell detachment and cell rounding through
phy) or an altered composition and a change in trypsin. The proteolytic function of trypsin forces
mechanical function, has remained unknown. Similar adherent cells to detach and to oat in suspension.
to collagen synthesis, there is an indication that protein Tendon broblasts in this state reveal a rounded
degradation is activated after exercise, in that local cellular shape and a loss of the elongated actin-based
levels of matrix metalloproteinases in tendon and laments (Fig. 2(ac)). Within 1 h after trypsination
muscle tissue are increased after acute exercise (Kos- and re-seeding, tendon broblasts adhere to culture
kinen et al. 2004). No good direct determination of plastic and begin to rebuild their characteristic mor-
collagen degradation has been provided so far, and phology, which is an elongated shape with long actin-
therefore the present data only indirectly point toward based cytoplasmic protrusions that interact through
an increased collagen turnover with exercise. It is actin-associated proteins with adjacent cells. In this
dicult to state how much of this increased collagen process, cells spread over the surface, form gradually
turnover is in fact turned into assembled load-bearing growing lamellipodia and establish multiple cell
collagen, but there is from data on connective tissue contacts (Figs 2(gi) and 3; Li et al., 2008). In tendon
in the lung reason to believe that at least a certain tissue, both adherens and gap junctions are found
percentage of the newly formed collagen will end up as (McNeilly et al., 1996; Banes et al., 1999; Ralphs et
insoluble collagen type I in the nal tendon structure al., 2002; Waggett et al., 2006; Richardson et al.,
(Laurent, 1987). 2007; Wall et al., 2007, unpublished observation).
Adherens junctions are cadherincatenin-mediated
cell adhesion sites. Cadherin molecules are transmem-
The tendon cell in humans: the indispensable cellular brane proteins and bind with their extracellular part
network of actin cytoskeleton and junctional through homophilic interactions to cadherins on
complexes neighboring cells. Ca21 ions are linked at the junctions
between cells, and thereby enable a rigid rod by
Clearly, the tendon broblast is considered a key player increasing stability to the adhesion site. On the in-
in tendon maintenance, adaptation to changes in home- tracellular side, cadherins are linked through catenin-b
ostasis and remodeling in case of minor or more severe and catenin-a to the actin cytoskeleton. During devel-
disturbances to tendon tissue. In postnatal tendons, the opment, cadherins play an essential role in a variety of
broblast is the major mechanoresponsive cell in the biological processes, such as cell sorting, cell polariza-
tissue (Yang et al., 2005). Although elaborate studies on tion, proliferation, apoptosis and tissue morphogen-
this cell type have been published during the last few esis. In postnatal tissue, adherens junctions control
years, the tendon broblasts have remained a some- cell polarity, cell migration and proliferation. Notably,
what under-researched topic, especially in postnatal cadherin-mediated adherens junctions are suggested
human tendon. To date, the human tendon broblasts to show a force-dependent behavior, and are likely
have been examined with regard to the cells role in the involved in mechanostransduction (Goodsell, 2002;
dierentiation potential (De Mos et al. 2007), the Pettitt, 2005; Ehrlich et al., 2006; Schwartz & DeSi-
existence and niche of tendon progenitor cells (Bi et mone, 2008).
al., 2007), expression patterns during in vitro culturing As mentioned above, cadherin molecules and the
(Almarza et al., 2008), cell morphology (Ujihara et al., actin cytoskeleton are linked together. The cytoplas-
2008), cell shape with regard to collagen expression (Li mic-located protein catenin-a binds to the actin cytos-
et al., 2008) and the capability of tendon broblasts to keleton; the co-localization of cadherin 11 and actin
form a cellular network in tendon (Wall et al., 2007). can be visualized on tendon broblasts (Fig. 3(f) and
Collagen brils are aligned in a well-organized (i)). During the process of contact formation, it seems
parallel order in tendon, and so are the cell-asso- as if the growing lamellipodia precedes the formation
ciated actin laments, following the crimp pattern of of adherens junctions, i.e. the elongation of actin-based

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Tendon adaptation to exercise

Fig. 2. Cellular morphology and Cad-


herin 11 localization in tendon bro-
blasts following detachment. (ac) Cells
in suspension: rounded cell morphology
with Cadherin 11 staining of the whole-
cell body. (df) 1 h postseeding: circular
cell morphology and prominent perinuc-
lear staining for Cadherin 11. (ge) 2 h
postseeding: Tendon broblasts start to
atten, and begin to form lammeliopo-
dia. (a), (d), (g): red color corresponds to
phalloidin staining; (b), (e), (h): green
color corresponds to Cadherin 11 stain-
ing; (c), (f), (i): computer-generated
merged images of the individually cap-
tured images; blue color corresponds to
nuclear stain with DAPI  40 magni-
cation.

Fig. 3. Cellular morphology and forma-


tion of adherens junctions in tendon
broblasts following detachment. (ac)
4 h postseeding: tendon broblasts with
expanding lamelliopodia, Cadherin 11
staining both perinuclear and localized
to the plasma membrane. (df) 8 h post-
seeding: broblasts with distinct actin
laments and formation of adherens
junctions (comb-like appearance). The
perinuclear localization is absent. (ge)
24 h postseeding: Fibroblasts with dis-
tinct actin laments and adherens junc-
tions with the typical comb-like
structure. The perinuclear localization is
absent. (a), (d), (g): red color corre-
sponds to phalloidin staining; (b), (e),
(h): green color corresponds to Cadherin
11 staining; (c), (f), (i): computer-gener-
ated merged images of the individually
captured images; blue color corresponds
to nuclear stain with DAPI. Scale
bar 5 50 mm,  40 magnication.

protrusions is observed when cadherin 11 primarily Ralphs and co-workers visualized the association of
appears perinuclear (Fig. 3(ac)). It could, however, cadherins and the actin cytoskeleton through co-
also be a simultaneous process of highly dynamically localization of N-cadherin and actin-based laments
expanding lamellipodia, cadherin adhesion formation in tissue obtained from chicken tendon. Recently,
in which the cadherin and catenin molecules enable we observed that cadherin 11 is present in human
further actin dynamics as a pushing force to estab- tendon tissue, and that tendon broblasts rapidly
lish multiple strong cell adhesions (Bryant and Stow, form cadherin-mediated adherens junctions in vitro
2004, Mege et al., 2006). to form a tight network of broblasts (Fig. 3).
While a detailed expression prole throughout Although it has not been suciently investigated,
tendon development and tissue maintenance is lack- the dynamic expression of dierent cadherin proteins
ing, two types of cadherins have been described, i.e. as a result of various environmental challenges could
N-cadherin and cadherin 11, both in embryonic and indicate a role of adherens junctions in physiological
postnatal tendon (Ralphs et al., 2002; Richardson and pathological adaptations in mature tendon (Fol-
et al., 2007; M. Bayer, unpublished observation). lonier et al., 2008).

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Kjr et al.
In contrast to adherens junctions (and tight junc- broblasts has several limitations, both with regard to
tions), gap junctions do not seal membrane-form ethical and methodological issues. However, opportu-
adjacent cells together, but build channels between nities in cell culture systems to mimic in vivo conditions
neighboring cells to allow shuing of small molecules, are considered as adequate tools for further research.
thereby enabling an active exchange through the inter- Based on existing knowledge of tendon broblasts,
cellular network. In postnatal tendon tissue, tendon new insights and the urgent need to enhance the results
broblasts express connexin 32 and connexin 43 and of the cells in healthy and diseased mature tendon, we
the junctional proteins are co-localized with actin. This suggest that understanding the cellular network and
was reported in avian as well as in human tendon (Wall the link between tendon broblasts with the matrix are
& Banes, 2005; Wall et al., 2007, unpublished observa- areas in tendon research that require profound inves-
tions). In postnatal tendon, age does not have a tigations in the nearer future.
signicant eect on the abundance of connexin expres-
sion; however, there is a distinct dierence between fetal
tendon and postnatal tissue. The amount of connexin Role of growth factors in tendon adaptation to
in the tendon during development is signicantly changes in loading
higher, at least in equine tendon (Stanley et al., 2007).
Tendons are constantly exposed to mechanical An important question in relation to increased col-
strain, and it is noteworthy that the actinconnexin lagen turnover is how tendon senses the external
co-localization is aected by mechanical loading, as loading during muscular contraction and more spe-
it was shown to increase concomitantly with an cically what factors are involved in this regulation.
increase of mechanical load. These ndings suggest The rst example of factors involved in collagen
a higher turnover of junctional proteins, or an synthesis regulation comes from the observation
increase in the formation of gap junctions between that apparently a gender dierence exists, in that
tendon broblasts exposed to strain, or an increased females respond less than males with regard to the
demand of stability of junctions between cells, increase in collagen formation after exercise (Miller
exposed to strain, or a combination of these. Block- et al., 2006a, b). The expected increase in collagen
ing gap junctions, on the other hand, resulted in a synthesis to exercise was less pronounced in women
decrease of collagen type I production, supporting than in males, and furthermore the basal collagen
that gap junctions play an indispensable role in synthesis rate was also lower in females compared
maintaining cellular function in tendon tissue (Banes with male counterparts (Miller et al., 2006a, b).
et al., 1999; Waggett et al., 2006; Wall et al., 2007). Further experiments in females who have varying
The importance of the actin cytoskeleton in pro- levels of sex hormones (e.g. oral contraceptives)
moting the formation of cell junctions is one of many suggest that estradiol may contribute to a diminished
indispensable roles of the lamentous network. collagen synthesis response in females (Hansen et al.,
Among various functions, one of them is the link 2008). Interestingly, this nding of a lower collagen
between the actin cytoskeleton and mechanotrans- synthesis rate both at rest and in response to exercise
duction. Although the activation of actin-linked in women vs males is correlated to a demonstration
signal transduction has not been studied suciently of lower stress to failure tolerance in patella tendons
in the tendon broblasts, investigations on other of females compared with males (Haraldsson et al.,
cell types show that broblasts sense, transduce 2005). Overall, this may contribute to our under-
and respond to changes in mechanical strain in the standing with regard to the fact that women experi-
environment through interaction with the matrix, ence a larger number of soft tissue injuries in e.g.
mainly through focal adhesion. Thereby, the cell cruciate ligaments than men, and may also be im-
creates a link through receptors on the cells with portant in understanding how rapidly humans of
ligands in the ECM and the actin cytoskeleton, and both genders adapt to not only physical training
enables adequate reactions through activation of but also how well they may resist prolonged periods
downstream eectors. The ndings by Ralphs et al. of reduced activity. The exact mechanism behind this
(2002) support a role of focal adhesions by reporting a gender-specic response is not denitively known,
co-localization of actin laments with the focal adhe- but the nding of a correlation between increased
sion protein vinculin in the tendon (Woods & Couch- estrogen levels and a reduction in collagen synthesis
man, 2001; Ralphs et al., 2002; Huveneers et al., 2008). in humans is supported by in vitro studies, where it
An increase in the knowledge of tendon broblasts has been shown that estradiol receptors are present in
in human tissue appears to be a necessity. It is ligaments (Sciore et al., 1998) and the nding that
important to obtain a comprehensive view of cellular estradiol per se can exert a collagen synthesis-inhibit-
processes in healthy tendon, to nd cues that might ing eect in tendons and ligaments (Liu et al., 1996;
play a critical role in diseased, degenerated and Yu et al., 2001). In addition to a suggested direct
injured tendon. The work on isolated human tendon inhibiting eect of estradiol upon collagen synthesis,

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Tendon adaptation to exercise
it may also be that estradiol levels exert an indirect (Kim et al., 2002; Nakatani et al., 2002) and patella
eect by inuencing other hormonal components tendon broblasts (Yang et al., 2004). Thus, these
of the human endocrine system. As an example, growth factors, especially TGF-b-1, may play an
more recently, high levels of circulating estradiol in essential role in mediating the adaptation response
women have been shown to be associated with low of tendon and muscle connective tissue to changes in
levels of circulating insulin-like growth factor (IGF-I), mechanical loading.
a substance that may be directly coupled to the degree We have recently investigated mRNA expression
of collagen synthesis increase with exercise. Thus, the of the stress-responsive growth factors, TGF-b-1,
gender dierences may not be an eect of estradiol IGF-I and CTGF, along with type I and III collagen,
directly, but rather an inuence of estradiol on IGF-I. in rat tendon and muscle after a short-term strength
An important regulating factor in collagen synth- training involving either pure shortening, lengthen-
esis is the growth hormone (GH)IGF-I axis, where ing or static contractions of plantar extensors (Hei-
in vitro data have shown a role for collagen forma- nemeier et al., 2007a, b). In the Achilles tendon, the
tion (Dssing & Kjr, 2005). As an example, it has expression of TGF-b-1 and IGF-I (but not CTGF)
been shown that IGF-I (and IGF-II) administration was increased in response to strength training, but no
in rabbits accelerates the protein synthesis in tendons dierence was seen between contraction types, even
(Abrahamsson, 1997), and likewise that the recovery though the force production was markedly higher
after tendon injury was accelerated when IGF-I was during lengthening contractions compared with
administered (Kurtz et al., 1999). Although GH in shortening contractions. In gastrocnemius muscle, a
skeletal muscle has been shown to exert an eect on similar regulation of gene expression was observed,
muscle growth in GH-decient individuals, the eect but, in contrast to the tendon response, the eect of
of GH supplementation on muscle protein synthesis lengthening contractions was signicantly greater
is absent in both young and elderly humans (Lange et than the eect of shortening contractions with regard
al., 2002). Despite this, it seems that GH/IGF-I to the expression of growth factors. Interestingly, the
inuences connective tissue, and administration of expression of type I and III collagen mRNA followed
GH over 3 weeks has been shown to elevate circula- a pattern very similar to that of TGF-b-1 and IGF-I
tion blood levels of procollagen propeptides (Long- expression in both tissue types. These results under-
obardi et al. 2000). Thus, it may be tempting to line that mechanical loading of tendon and muscle
speculate that GH/IGF-I has a stimulating eect on induces collagen expression, and they support a role
tendon tissue. In dwarf rats, GH administration has for TGF-b-1 and IGF-I as mediators of this eect.
been shown to increase the expression of both collagen Importantly, it is also indicated that tendon tissue,
type I and III in intramuscular broblasts (Wilson et although it reacts to loading, is less sensitive to
al., 1995). Recently, it has been shown that IGF-I is dierences in the mechanical stimulus compared
present in human Achilles tendon linked directly to with skeletal muscle.
broblasts, and furthermore a detectable interstitial With the eect of loading on tendon and muscle
concentration has been demonstrated in human ten- connective tissue in mind, it is relevant to consider
don (Olesen et al., 2006). Although this supports the whether unloading will lead to an opposite response
possibility that IGF-I may play a role in human such as a decrease in the expression of collagen and
tendon, the following question, however, remains: to collagen-inducing growth factors (De Boer et al.,
what extent is IGF-I upregulated with exercise? 2007). Additionally, it would be interesting to inves-
Induction of collagen expression in response to tigate whether tendon, also in an unloading situation,
increased loading is seen in many cell and tissue will react less markedly than muscle. To study the
types, and has been suggested to depend on a coordinated response of tendon and muscle to un-
mechanically induced expression/secretion of col- loading, we measured tissue mass, as well as mRNA
lagen-inducing growth factors. These growth factors expression levels for collagen and growth factors, of
are then thought to work in an auto/paracrine manner rat Achilles tendon and soleus muscle after 7 and 14
to induce ECM protein production (Sarasa-Renedo & days of hindlimb suspension. In accordance with
Chiquet, 2005). Several growth factors, which stimu- earlier studies, the soleus muscle mass decreased to
late collagen synthesis, are expressed in response to 50% of the control levels after 14 days of unloading
mechanical loading. These include transforming (Hirose et al., 2007). Achilles tendon mass, on the
growth factor-b-1 (TGF-b-1) (Schild & Trueb, 2002), other hand, was not aected by suspension, and
connective tissue growth factor (CTGF) (Frazier et al., although this may seem surprising, previous studies
1996; Schild & Trueb, 2002) and IGF-I (Hansson et al., have shown unchanged cross-sectional areas of rat
1988; Abrahamsson & Lohmander, 1996; Butt & Achilles tendons after several weeks of unloading in
Bishop, 1997). Importantly, loading-induced type I both human and animal studies (Bikle et al., 1994;
and/or type III-collagen expression appear to depend Almeida-Silveira et al. 2000; Matsumoto et al., 2003).
directly on TGF-b-1 activity in human ligaments With regard to gene expression, the response to

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Kjr et al.
unloading was clearly not opposite to that of loading. tendon cross-sectional area, which appears to be
The expression of collagen I and III, TGF-b-1 and site specic (Rosager et al., 2002; Magnusson &
CTGF was not aected in either tendon or muscle by Kjr, 2003; Kongsgaard et al., 2005). However, in
hindlimb suspension (although collagen III tended to an intervention study it was shown that 9 months of
decrease in muscle after 7 days). Furthermore, we endurance training in untrained persons left the
found an increased expression level of IGF-I in tendon Achilles tendon CSA unchanged (Hansen et al.,
tissue in response to unloading. This was perhaps 2003). On the other hand, animal data have shown
unexpected, but a similar eect of unloading has that muscle strength/size is related to the tendon size
been shown in bone tissue, and the IGF-I mRNA (Elliott, 1965), suggesting that perhaps the magni-
increase may represent a secondary response to an tude of loading inuences tendon size. In animal
unloading-induced IGF-I resistance (Bikle et al., 1994; studies, the cross-sectional area of tendon often
Sakata et al., 2004). adapts dierentially between tendon types and thus
With return to normal cage activity (reload) after indicates that responses may be tendon specic.
the hindlimb suspension period, the skeletal muscle Several human studies have shown that resistance
expression of collagen I and III was markedly in- training over 1214 weeks that produces increases in
duced (410-fold), while the expression of growth muscle strength of up to 21% does not result in an
factors was moderately induced. This indicates an accompanying increase in tendon CSA (Reeves et al.
overall anabolic response in the muscle tissue, which 2003a, b; Kubo et al., 2006), but rather a markedly
is a logical response when considering the marked altered modulus, which implies that there is a change
loss of soleus muscle mass seen after suspension in the composition of the structure rather than the
(50% at day 14 HS) compared with the moderate size. In contrast, we recently showed that, in humans,
loss of body mass. In tendon tissue, the response to resistance training for 12 weeks yielded region-spe-
reload was far less pronounced, and the expression of cic increases in PT CSA, without a change in
growth factors corresponded to control levels during modulus (Kongsgaard et al., 2007), which was the
the entire reload period. The moderate response to rst human intervention study to report such tendon
reload supports that the changes induced by suspen- hypertrophy. It is possible that previous studies have
sion were limited in tendon, compared with muscle, overlooked the tendon hypertrophy because it occurs
as indicated by the stable tissue mass and the un- in the distal and proximal end of the tendon and not
changed expression of matrix-related genes during in the middle, where it is commonly measured. We
unloading. Meanwhile, the expression of both col- subsequently followed up on these ndings with an
lagen I and III was actually induced in tendon after alternative design: cross-sectional studies are ob-
4 days of reload, indicating that some changes must viously limited due to issues of training history,
have taken place in the tendon tissue during the selection bias and inter-subject variations. Longitu-
suspension period. This is supported by previous dinal training studies may be of insucient duration
results showing a pronounced disuse-induced change to produce a robust tendon hypertrophy response,
in tendon mechanical properties (Almeida-Silveira and the existing training studies have examined
et al. 2000; Matsumoto et al., 2003), and it could be tendon size in a region that appears to be unrespon-
speculated that changes in the tendon matrix induced sive to training-associated adaptation. Some of these
by unloading will aect the tendoncell response to limitations may be partially overcome by examina-
the resumption of a normal loading pattern and thus tion of region-specic PT properties in persons who
lead to increased collagen expression. engage in sport where one lower extremity is habi-
In conclusion, the general response of both tendon tually subjected to more loading than the contral-
and muscle tissue to unloading does not appear to ateral (control) side, such as in fencing or badminton.
follow a pattern opposite to that of a loading res- When we examined patellar tendon size and mechan-
ponse, and in general the tendon response appears to ical properties in subjects who display a side-to-side
be less pronounced than the muscle response. This strength dierence of  15% due to persistent sport-
could indicate that tendon tissue is protected from induced loading over several years, we found a
rapid changes in tissue mass, while muscle, which is regional variation in CSA along the patellar tendon,
known to act as a protein store for the organism, is which markedly inuenced average stress along the
subject to substantial and fast changes in tissue mass. tendon (Couppe et al., 2008). The habitually more
loaded tendon of the stronger extremity had a greater
cross-sectional area compared with the contralateral
Effect of loading on the properties of the human side. The lead extremity also displayed greater stiness
patellar tendon in vivo than the contralateral side, while the modulus did not
dier signicantly. In sum, these data show that a
In humans, cross-sectional data suggest that endur- habitual loading is associated with a robust change of
ance training is associated with a larger Achilles the patellar tendon size and mechanical properties.

506
Tendon adaptation to exercise
Damage and healing of tendon recovery and eect of eccentric rehabilitation. A few studies have
rehabilitation of human tendon suggested potential structural, functional and mole-
cular explanations (Ohberg & Alfredson, 2004; Oh-
Overuse injuries of tendons are a highly frequent and berg et al., 2004; Langberg et al., 2007; Rees et al.,
disabling condition aecting both professional and 2008). After tendon injury, most repair activity is
recreational athletes as well as workplace employees associated with cells from the epitenon and endote-
undertaking forceful and repetitive tasks (Langberg non migrating to the lesion and synthesizing a new
& Kongsgaard, 2008; Tan & Chan, 2008). The pre- matrix (Jones et al., 2003; Kajikawa et al., 2007).
valence of tendinopathies has been reported to range This migration and matrix synthesis may also be
from 2% among the employment-active part of the stimulated by eccentric loading as controlled ec-
population in western countries to as high as 55% centric rehabilitation exercises have been found to
among certain jumping athletes (Lian et al., 2006). lead to increased collagen synthesis in injured ten-
Tendinopathy can occur in almost any tendon. dons only, in contrast to the healthy leg (Langberg et
Common examples include Achilles tendinitis, patellar al., 2007). This increase in collagen synthesis is
tendinitis, tennis elbow, golfers elbow and supraspi- correlated with a decrease in pain during activity,
natus tendinitis. The high prevalence, together with the indicating that the condition emerged from a mis-
fact that the pathologies often become chronic, makes match between the tissue strength and the load
tendinopathies a large socioeconomic problem. In spite during activity (Langberg et al., 2007). Alternatively,
of the extremely high prevalence of tendinopathies, the rehabilitation training resulted in a reduced
knowledge regarding eective rehabilitation regimes release of pain-inducing factors. In a recent study
and medical treatments is limited (Langberg & Kongs- on the eect of experimental induced pain (injections
gaard, 2008). One of the reasons is the lack of knowl- of hypertonic saline in the Achilles tendon), we
edge regarding the pathogenesis of the tendinopathies. showed that the muscular control of the calf muscle
Activity-related pain, focal tendon tenderness and is disturbed as a result of the pain (M. Henriksen
decreased strength and movement in the aected area et al., unpublished observation). When loaded eccen-
characterize tendinopathy. Previously, it was be- trically, the pain leads to increased physiological
lieved that inammation was the key contributor to tremors not present during concentric loading. This
tendon overuse problems, but, although still debated, could indicate that the broblasts migrating into the
there is growing evidence suggesting this is not the injured tissue (Jones et al., 2003) are exposed to a
case (Movin et al., 1997; Alfredson et al., 1999). At dierent load pattern when subjected to eccentric
present, tendinopatheis are classied as a primarily contraction, leading to an increase in collagen synth-
degenerative condition with histologic examination esis and thus stimulating the healing of the injured
of tendinosis tissues showing a disrupted collagen tissue. However, despite this sparse information, the
matrix, increased cellularity and proteoglycan con- number of unanswered questions are still substantial,
centration, but lack of inammatory cell inltration and much more research is needed before the etiol-
(Fu et al., 2002; Riley, 2008). Unpublished data, ogy of tendinopathy is understood and the correct
though, indicate that the strength-bearing structures, rehabilitation is determined.
the brils, are unchanged to normal conditions in
diameter and distribution in chronic overuse condi-
tions (J. Pingel, unpublished observation), suggesting
that the problem is mainly situated between the Perspectives
brils. Multiple studies have demonstrated neovas-
cularity in the overused tendons (Ohberg et al., 2004) The increased knowledge of the collagen response of
along with ingrowths of nerve endings. In spite of human tendon toward loading is important for
these observations, the molecular and biomechanical recommendations in training and rehabilitation regi-
mechanisms underlying the cause and progression of mens in order to maximize eect without overloading
tendinopathy as well as the healing of the connective tendons. It is also important for the understanding of
tissue are not understood (Riley, 2008). long-term tissue changes in relation to chronic train-
During the last decade, treatment involving heavy- ing. Clearly, it represents only one side of the coin,
load eccentric training has been shown to provide and studies on other matrix proteins and structures
good clinical results in the treatment of both Achilles (e.g. cross bindings) will be important to explain the
tendinosis (Alfredson et al., 1998; Langberg et al., conversion of biochemical changes in tendon into
2007) as well as in the case of patella tendinopathy structural and functional adaptations.
(Jonsson & Alfredson, 2005; Kongsgaard et al., 2006;
Frohm et al., 2007). At present, not much is known Key words: broblast, growth factors, exercise, physi-
about the mechanism behind this positive clinical cal training.

507
Kjr et al.
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