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Sports Medicine Research Unit, Department of Rheumatology, Copenhagen University Hospital at Bispebjerg, Copenhagen, Denmark
ABSTRACT I. INTRODUCTION II. CONVERSION OF MECHANICAL LOADING INTO TISSUE ADAPTATION OF TENDON AND EXTRACELLULAR MATRIX OF SKELETAL MUSCLE: THE GENERAL CONCEPT III. TENDON AND SKELETAL MUSCLE EXTRACELLULAR MATRIX CONTENT: ORGANIZATION AND PHYSIOLOGICAL FUNCTION A. Tendon Components B. Tendon Fibroblast Signaling C. Tendon Vasculature and Blood Flow Regulation D. ECM Components in Skeletal Muscle E. Functional Implications of ECM in Tendon and Muscle IV. REGULATION OF COLLAGEN AND OTHER EXTRACELLULAR MATRIX PROTEIN SYNTHESIS: INFLUENCE OF CHANGES IN MECHANICAL LOADING A. Steps of Collagen Synthesis: Methodological Considerations B. Determination of Collagen Turnover in Humans C. Responses to Increased Loading: Acute and Chronic Exercise D. Immobilization and Collagen Turnover V. DEGRADATION OF CONNECTIVE TISSUE IN TENDON AND SKELETAL MUSCLE: EFFECTS OF CHANGES IN MECHANICAL LOADING A. MMPs B. TIMPs VI. STRUCTURE OF EXTRACELLULAR MATRIX IN TENDON AND MUSCLE: RELATION TO MECHANICAL AND VISCOELASTIC PROPERTIES A. Extensibility of Tendons B. Repetitive Loading and Tendon Properties C. Aging, Disuse, and ECM D. Integrated Muscle-Tendon ECM Properties VII. LOADING AND OVERLOADING OF CONNECTIVE TISSUE STRUCTURES IN TENDON AND SKELETAL MUSCLE: ROLE OF GROWTH FACTORS A. TGF-{beta} and CTGF B. FGF C. IL-1 and IL-6 D. IGF and IGF-Binding Proteins VIII. COUPLING OF REGULATORY PATHWAYS FOR EXTRACELLULAR MATRIX TURNOVER AND SKELETAL MUSCLE CELLS TO MECHANICAL TISSUE LOADING A. Development of Skeletal Muscle and Intramuscular ECM B. ECM and Skeletal Muscle Interplay in Mature Tissue IX. CLINICAL PERSPECTIVES: PHYSIOLOGICAL UNDERSTANDING OF TISSUE OVERUSE A. Tendon Overuse B. Development of Tendon Injury: Predisposing Factors C. Tendon Rupture: Preceding Overuse D. Experimental Tendon-Overuse Models E. Tendon Overuse and Inflammation F. Tendon Overuse, Blood Flow, and Tissue Oxygenation G. Tendon Injury and Pain H. Perspectives for Treatment X. FUTURE PERSPECTIVES XI. SUMMARY
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I. INTRODUCTION |
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The ECM consists of a variety of substances, of which collagen fibrils and proteoglycans are truly ubiquitous (153). In addition to the proteoglycans (PG), the hydrophilic ECM includes (164, 339, 581) a variety of other proteins such as noncollagen glycoproteins (582, 583). It is known that the force transmission of the muscle-tendon complex is dependent on the structural integrity between individual muscle fibers and the ECM (48) as well as the fibrillar arrangement of the tendon and its allowance for absorption and loading of energy (15, 16). Furthermore, it is well described that the tensile strength of the matrix is based on intra- and intermolecular cross-links, the orientation, density, and length of both the collagen fibrils and fibers (57, 447, 496, 497, 630–632, 640). However, the signals triggering the connective tissue cells in response to mechanical loading, and the subsequent expression and synthesis of specific extracellular matrix proteins, as well as its coupling to the mechanical function of the tissue are only partly described (51–53, 173, 181).
This review focuses on the physiological role of the ECM, especially collagen, for the tendon-muscle interaction and the adaptation to mechanical loading. Somewhat in contrast to the classical view of the ECM tissue being relatively static and inert, evidence is evolving that tendons and intracellular connective tissue are more dynamic structures that adapt to the variety of functional demands that the musculoskeletal system is subjected to, and that this tissue adapts both in a structural and functional way to mechanical loading (53, 173, 386, 630). Recent development of refined in vivo techniques have underlined that connective tissue of skeletal muscle and tendon is a lively structure with a dynamic protein turnover and that it possesses the capacity to adapt greatly to changes in the external environment such as mechanical loading or inactivity and disuse.
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II. CONVERSION OF MECHANICAL LOADING INTO TISSUE ADAPTATION OF TENDON AND EXTRACELLULAR MATRIX OF SKELETAL MUSCLE: THE GENERAL CONCEPT |
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The ECM is a conglomerate of
substances in which biochemical and biophysical properties allow for
the construction of a flexible network that integrates
information from loading and converts it into mechanical capacities
(152,
494,
690).
It serves as a scaffold for adhesion of
cells mediated by integrins, dystroglycan, and proteoglycans at the
cell surface and of tyrosine kinase receptors (98,
290).
The interaction between the ECM and the adhesion molecules leads to
activation of intracellular signaling pathways and
cytoskeletal rearrangement (52,
82,
114).
In combination with this, the PGs with their glycosaminoglycan side
chains are able to bind and present growth factors to their
relevant receptors, and furthermore, the ECM can release growth
factors upon relevant mechanical stimulation. The complete
signaling pathways responsible for mechanotransduction responses are
yet to be described, but several candidates have been suggested
from investigations on a variety of
fibroblasts in dermis, vasculature, and cardiac muscle (166,
396,
667).
Integrin molecules are major structural components of
adhesion complexes at the cell membrane linking the ECM to the
cytoskeleton (108,
128,
541).
In this way integrins establish a mechanical continuum along which
forces can be transmitted from the outside to the inside
of the cell, and vice versa (238,
290,
291,
667,
668).
It is believed that integrins are the sensors of
tensile strain at the cell surface (290).
Ingber et al. (291)
have suggested that integrins together with the cytoskeleton form a
mechanically sensitive organelle. At the myotendinous junction, lack
of integrin expression will lead to
structural damage during muscle contraction (442).
Integrins are important structural components of the
adhesion complexes at the cell membrane, and they play a crucial
role in linking the ECM to the
cytoskeleton (227,
412,
418,
419,
579).
Thereby they provide a bridge through which forces can be
transmitted between inside and outside of the
cells in a two-way street principle. Further evidence for this is
provided by the fact that integrins can convert mechanical signals to
adaptive responses in the cell (115,
591).
In addition to integrins, also the dystrophin-glycoprotein complex
plays an important role in mechanotransduction of
muscle and tendon tissue (107,
130,
288).
The  649 -- Physiological Reviews_files/beta.gif)
-subunit cytoplasmic domain of
integrin is interacting with the cytoskeleton, and the demonstration
of  649 -- Physiological Reviews_files/alpha.gif)
71-integrin linked to laminin in the ECM is
important for signal transduction (81,
309a,
309b),
and lack of the 2-laminin leads to muscle dystrophy. Interestingly,
overexpression of
71-integrin in dystrophin-deficient mice leads to
reduction in dystrophic symptoms, indicating that some substitution
effects exist between integrin and laminin (107).
Extracellular matrix ligands for integrins are known
to be collagens, fibronectin, tenascin, and laminin (412).
Several studies have demonstrated that the expression of
several other ECM components are controlled by the level
of mechanical loading. For example, collagen
XII and tenascin-C, which are present in both tendon and other
connective tissue structures like ligaments, have been shown to
increase their expression and synthesis when fibroblasts are
stretched in vitro and are suppressed in cells that are left in a
relaxed state (127,
129).
Although not yet confirmed, integrins are likely candidates for
sensing tensile stress at the cell surface (290,
683,
694,
698).
Thus some evidence indicates that integrin-associated proteins are
involved in the signaling adaptive cellular responses to mechanical
loading of the tissue, and it is likely that this
takes also place in tendon and skeletal muscle ECM-related
fibroblasts (531).
Several intracellular pathways for mechanotransduction signaling have been suggested, including focal adhesion kinase (FAK), paxillin, integrin-linked kinase (ILK-1), and mitogen-activated protein kinase (MAPK) (127, 206, 207, 241, 451, 618). MAPK is crucial for the conversion of mechanical load to tissue adaptation inducing signaling from the cytosol to the nucleus. It is well described that several cell types and subsets of MAPKs such as extracellular signal-regulated kinase 1 and 2 (MAPK-erk1+2, p44), stress-activated protein kinases p38 (MAPK-p38), c-jun NH2-terminal kinase (MAPK-jnk, p54), and extracellular signal-regulated kinase 5 (MAPK-erk5) can be activated by mechanical stress, as well as by lowered pH, growth factors, hormones, and reactive oxygen species (250, 414, 678, 693, 696). In regard to mechanical loading, it has been shown in muscle that MAPK can be activated both as a result of active muscle contraction (36, 37, 559) and after passive stretch (161, 439). The activation of MAPK results not only in a production of transcription factors, thus mediating gene expression, but also in an activation of the protein synthesis on the translational level through eukaryotic initiation and elongation factors (229). It has furthermore been suggested that the mode of mechanical load is coupled to a certain type of MAPK activation. In line with this, it has recently been shown in rat skeletal muscle cells that concentric activation of muscle associated with metabolic and ionic changes resulted in a preferential increase in MAPK-erk1+2, whereas intense eccentric tensile loading with barely any metabolic changes resulted in a marked increase in MAPK-p38 (as well as in MAPK-erk 1+2) (693). In another study that also used rat skeletal muscle, a strong relationship was found between peak tension (whether active and/or passive) and MAPK-jnk (439). This falls in line with the demonstration of MAPK-jnk activation and the induction of immediate early genes by mechanical stress in smooth muscle cells (253). Whether any marked increase in MAPK-p38 is found in skeletal muscle is still debatable. Whereas one study could not find any increase in MAPK-p38 in rat muscle during concentric, stretch, or eccentric muscle activity (439), another study found a marginal and late increase in MAPK-p38 (37, 241) while a third study found MAPK-p38 activation in exercised human skeletal muscle (674). It has been shown that activation in skeletal muscle of MAPK-p38 is fiber-type specific (243). Somewhat in contrast to muscle, it seems very clear that in connective tissue MAPK-p38 is mainly activated with mechanical stretching of the tissue (127). Findings on regulation of matrix metalloproteinase (MMP) activation in fibroblasts point toward a differentiated interplay between MAPKs, in which MAPK-p38 is important for induction of MMP, while MAPK-erk1+2 mediates the repression (534). Although not conclusive, these findings are compatible with stress pattern-dependent MAPK pathways in both the muscle cell and the fibroblast and suggest an intimate interplay between the muscle cell and intramuscular connective tissue in response to mechanical loading. Evidently, such pathways do not in any way rule out other mechanistic pathways (e.g., calcium-dependent pathways) to be involved in mechanotransduction also (36).
Taken together, it is likely that separate modes of tissue loading and thereby of physical training will differentially stimulate the subtypes of MAPK in both myocytes and fibroblasts. Most likely, endurance like oxidative loading of tissue stimulates MAPK-erk, whereas strength type of exercise is more likely to use the MAPK-jnk pathway (693). Furthermore, passive stretch both in muscle and connective tissue preferentially gives rise to stimulation of MAPK-p38 (89). In human models, stretch does not cause any increased muscle protein synthesis, and thus does not result in any hypertrophic effect on the muscle (211). The fact that the stretch of muscle cells and of fibroblast shows parallel MAPK activation suggests that adaptive processes in intramuscular connective tissue interact closely with those of skeletal muscle tissue when subjected to mechanical loading.
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III. TENDON AND SKELETAL MUSCLE EXTRACELLULAR MATRIX CONTENT: ORGANIZATION AND PHYSIOLOGICAL FUNCTION |
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Tendons consist of a systematic and densely packed
organization of
connective tissue dominated by collagen organized into fibrils,
fibers, fiber bundles, and fascicles, as well as by the presence
of other ECM proteins. The nature of the
individual components of the
tendon is equipped to withstand high tensile forces (224,
627,
632).
The division of tendons into fibrils ensures that
minor damage does not necessarily spread to the entire tendon,
and also provides a high total structural strength (Fig.
1). Tendon consists of
55–70% water, and a substantial part of
this is associated with proteoglycans in the ECM (187,
307,
546,
659,
660).
Of the tendon dry weight, 60–85% is collagen.
This collagen is predominantly type I ( 649 -- Physiological Reviews_files/sim.gif)
60%) arranged in tensile-resistant fibers, and composed
of two 1- and one 2-chains. These are products of
separate genes rather than a posttranslational modification
of a single molecule. Also, collagen types III
(reported between 0 and 10%), IV (2%) (12,
260),
V, and VI are present (72,
74,
307,
324,
652,
653).
In addition to this, a small amount of elastin fibers are present (2% of dry weight) (194,
195,
307).
Apart from a very small amount of
inorganic substance (<0.2%), the remaining substance consists
of different proteins (accounting for
4.5%) (660),
but very little information is present as to the relative
contribution of these (10b).
It has been shown that the inorganic substance is dominated by PGs,
especially small leucine-rich proteins of
which decorin (up to 1%) (164,
307,
660)
and cartilage oligomeric matrix
protein (COMP, up to 1%) (465,
601,
604)
are probably the most abundant. In addition, other small leucine-rich
PGs such as fibromodulin, biglycan (up to 0.5%), and lumican,
together with osteoadherin, tenascin-C, proline argininerich end
leucine-rich repeat protein, optican, keratocan, epiphycan, syndican,
perlecan, agrin, fibronectin, laminin, vercican, and aggrecan are
present in tendon tissue (300,
307,
537).
The PGs and water are thought to have a spacing and lubricating
role for the tendon, whereas the role
for several of the small and nonaggregating leucinerich PGs is
more unclear. The proteoglycans also seem to play an important
role in fibril fusion, as do fibrillin
molecules aligning along fibrils (50).
Tendons vary markedly in design, most likely coupled to their
function. In the quadriceps the tendon can be found to be short
and thick, whereas several of the
tendons to the fingers or toes are long and thin. Furthermore,
tendons may vary in thickness along its length and are often
surrounded by loose connective tissue lined with synovial cells, the
paratenon, to allow for large movements of the
tendon. The epitenon is the connective tissue sheet that immediately
surrounds the tendon, and it consists of
loose, fatty, areolar tissue that allows for the tendon together with
the tendon sheet, the peritendon, to glide against adjacent tissue
(574).
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B. Tendon Fibroblast Signaling
Tendons are dominated by fibroblasts. In addition, also other cell types like endothelial cells and mast cells as well as axons are, together with the ECM, also present in tendons. It has been demonstrated that tendon fibroblasts lie in longitudinal rows and have numerous sheet-like cell extensions that extend far into the ECM (447) (Fig. 1). Isolated tendon fibroblasts respond to mechanically induced loading with expression of several ECM components (53). In the intact tendon, cells are linked to each other via gap junctions as evidenced by immunolabeling for connexin32 and connexin43 (447, 528). Where the latter represents the meeting of cell processes as well as where cell bodies meet, the former only represents contact between cell bodies. In total, the architecture of the fibroblasts of the tendon and their interconnection provides a three-dimensional network that surrounds the collagen fibrils and provides a basis for cell-to-cell interaction. In vitro, tendon cells upregulate collagen and gap junction production under mechanical cyclic loading, and pharmacological inhibition of the gap junction leads to loss of this response (52, 662). Gap junctions must under loading be able to withstand high loads and have been shown to be coupled to the actin cytoskeleton (394, 395, 695).
In articular chondrocytes and compressed tendon regions, a
compression-sensitive organization of
intermediate filaments has been shown (179,
528,
529).
As well actin filaments and fibers have been shown in developing
intervertebral discs and in scar connective tissue (194).
However, the demonstration of
these has not been put into a functional perspective (331,
469).
It has been demonstrated that in knockout mice for the intermediate
filament vimentin, -smooth muscle actin organization is abnormal in dermal
fibroblasts and that their contractile ability is impaired (180).
Recently, it has been shown that tendons have actin-based cell-cell
interaction (515)
and that actin stress fibers run along the rows of
fibroblasts (529).
When mechanically loaded, junctional components n-cadherin and
vinculin rose together with tropomyosin, without any change in actin
levels. The rise in cadherin and vinculin suggests an increased
cell-cell adhesion or cell-matrix
adhesion. This suggests that mechanical load transforms fibers into
partly contractile components that may contribute to an active
mechanism in the recovery after stretch and that these structures
can maintain the integrity of the
longitudinal tendon rows and to monitor tensile load and contribute
in the mechanotransduction during exercise (529).
C. Tendon Vasculature and Blood Flow Regulation
Compared with muscle, tendons have relatively limited vasculature,
and the area occupied by vessels represents 1–2% of the entire ECM (373,
374).
The vessels mainly emanate from the epitenon where longitudinal
vessels run into the endotenon (10c,
341,
373,
374).
Supplying arteries and arterioles may come from the perimysium at the
musculotendinous junction and vessels from the tendon bone junction
(113,
137,
574).
Long tendons are supplied by several vessels along their length (271,
341).
Due to the large excursion (up to 6 cm) that some tendons experience
during movement, the vessels to such tendons need to be long and
often winding in nature.
The ECM in relation to both muscle and tendon is extensively filled with blood vessels (508, 509), to provide the contracting muscle with oxygen and substrate for energy production, and to ensure an efflux from musculature of combustion products. It remains, however, unsolved to what extent the blood flow to connective tissue alters with mechanical loading of the tissue. In the resting state, rabbit tendons have been shown to have tendon flow of around one-third of that in muscle, and it is known that blood flow in both tendons and ligaments increase with exercise and during healing in animals (46). Both with the use of radiolabeled xenon washout technique from peritendon tissue as well as with application of near-infrared spectroscopy and simultaneous infusion of contrast substance (95), it has been possible to demonstrate in human models that blood flow within and around tendon connective tissue increases up to sevenfold during exercise, both in young, middle-aged, and elderly individuals (93, 94, 377, 378, 381). This increase is by far smaller that the 20-fold increase in adjacent skeletal muscle blood flow under similar exercise conditions (93, 94). However, compared with the metabolic activity of the tendon during exercise, it might be adequate. Furthermore, it can be shown that skeletal muscle blood flow during maximal exercise is close to what is possible to achieve with postocclusion reactive hyperemia, while the flow in tendon is still only 20% of that during maximal exercise (93, 94). This implies that tendon flow is not simply a function of skeletal muscle blood flow and that its regulation represents a separate regulatory system.
Vasodilatory agents have been measured simultaneously in skeletal muscle and its adjacent tendon during mechanical loading in vivo, and it has been found that adenosine concentrations rise in an intensity-dependent fashion in muscle, whereas the changes in tendon were less marked and unrelated to intensity (375). Furthermore, bradykinin concentrations rose in parallel in the two tissues during exercise, and already elicited its maximal response at low exercise loads (375). The changes in tissue bradykinin concentrations are in the range that has been found to cause a vasodilatory effect on endothelium (554). These findings indicate that these two substances are involved in blood flow regulation in skeletal muscle and tendon with exercise and that bradykinin is involved in the blood flow increase during lower work loads both in tendon and muscle. Whether bradykinin exerted its vasodilatory effect directly on the vasculature (109) or more indirectly via release of other substances as nitric oxide (NO) (490), prostaglandins (58), or endothelium-derived hyperpolarizing factor (EDHF) (279, 458) is yet to be established.
Interestingly, it has been shown that prostaglandin concentrations rise both in muscle (214, 317) and in connective peritendinous tissue (385) with exercise. Whereas inhibition of prostaglandin synthesis by itself did not inhibit total flow during exercise in skeletal muscle, but did so only if simultaneous blockade also of NO synthesis was performed (96), the peritendinous and tendinous blood flow during exercise was diminished by 40–50% compared with control exercise without blockade (376). This differentiated regulation of blood flow regulation in skeletal muscle and tendon tissue, respectively, can be hypothesized to imply also a differentiated regulation of blood flow within the skeletal muscle itself. This would be so if parts of the vasculature in muscle is located in regions with abundance of ECM, i.e., aponeurosis and perimysial tissue. The finding of flow heterogeneity within skeletal muscle as well as the demonstration of nutritive and nonnutritive vessels in skeletal muscle (94, 95, 137) are certainly supporting evidence for such an idea. It would also explain a separate role during exercise for vessels that were very responsive to vasodilation dependent on work load and thus providing maximal supplementation of substrate and oxygen to the muscle, and on the other hand vasculature located in connective tissue, both within the muscle and in relation to tendon tissue, where flow is coupled to inflammatory activity in repair processes for the ECM. The latter would also serve as a kind of shunt with the potential of partly limiting its vasodilation to share blood with the nutritive vessels during exercise.
With regard to the ECM, the main question remains whether the increase in flow is sufficient to meet the oxidative needs of the tendon and its cells during exercise. Determination of oxygen saturation and content of the Achilles tendon region in humans has been performed using near-infrared spectroscopy with the addition of a dye dilution method (94–96). When simultaneous recording of tissue oxygenation and blood flow of human tendon regions was performed both at rest and during muscular contractions, it can be demonstrated that a tight correlation exists between increasing blood flow and declining oxygen tissue saturation (334) (Fig. 2). This correlation could indicate a coupling and fits very well with what is found in skeletal muscle during exercise (96). This illustrates that during exercise the estimated oxygen uptake in humans tendon regions rises severalfold compared with the resting state and that even during intense mechanical loading of tendons, there is no indication of any tissue ischemia.
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D. ECM Components in Skeletal Muscle
Intramuscular connective tissue has multiple functions (301a, 407) (Fig. 3). First it provides a basic mechanical support for vessels and nerves. Second, the connective tissue ensures the passive elastic response of muscle. Third, it is now clear that force transmission from the muscle fibers not only is transformed to tendon and subsequent bone via the myotendinous junctions but also via lateral transmission between neighboring fibers and fascicles within a muscle (228, 338, 415, 626). It has been shown that tension developed in one muscle part can be transmitted via shear links to other parts of the muscle, and that even the cutting of an aponeurosis in a pennate muscle still maintains much of the force transmission (401). The perimysium is especially capable of transmitting tensile force (631). Although studies have also demonstrated a potential of the endomysium for force transmission, the orientation and curvilinearity of the collagen fibers provide high amounts of elasticity and thus not sufficient stiffness to function optimally as a force transmitter.
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Intramuscular connective tissue accounts for 1–10% of the skeletal muscle and varies quite
substantially between muscles (208,
301,
391,
628)
(Fig.
3). Whereas the endomysium encloses each individual muscle fiber
with random arrangement of
collagen fibrils to allow for movement during contraction, the
multisheet-layered perimysium runs transversely to fibers and holds
groups of fibers in place, while the epimysium
is formed of two layers of
wavy collagen fibrils to form a sheetlike structure at the
surface of the tendon. It has been demonstrated in bovine
muscles that the endomysial content can vary between 0.5 and 1.2% of the muscle dry weight, whereas the
perimysium accounts for between 0.4 and 4.8% (520).
This relatively small variation in the endomysial compared with
perimysial connective tissue content between muscles could indicate
that at least some functional differences between muscle groups
related to connective tissue content are mainly defined by perimysial
characteristics. The intramuscular connective tissue is dominated by
collagen and ensures not only an organization into fasicles and
fibers, but contributes importantly to the force transmission (39).
Several collagen types have been identified in intramuscular
connective tissue (up to 7) (174,
410,
411),
and whereas type IV dominates the basement membrane adjacent to
the plasma membrane of the sarcolemma (12,
342),
the fibrillar collagen type I and III (and to some extent type V)
dominates the epi-, peri-, and endomysium (the reticular layer). By
far type I collagen dominates the intramuscular collagen content
(reported from 30% and up to 97% of
total collagen) (47,
48,
257,
407).
At the other end of the scale, collagen types II, VI,
IX, XI-XVI, and XVIII-XIX represent only very minor amounts (88a,
167,
252a,
410,
411,
440,
471,
486).
It is likely that the difference in relative content of
connective tissue in specific muscles is coupled to function and the
role of
connective tissue (247,
248).
Differences between muscles with regard to their relative content and
type of collagen is already present early in
development (483,
485),
and in cattle, the concentration of
hydroxyproline as well as of
collagen type I and III achieve their highest levels two-thirds
through gestation (411).
Interestingly, the highest collagen concentrations are achieved at
the time when myotubes undergo their first phase of
morphological and contractile differentiation (411).
Furthermore, small leucine-rich proteoglycans of
intramuscular connective tissue are expressed in parallel with
development of skeletal muscle (506).
Decorin and fibromodulin mRNA were markedly elevated for a few
days, and biglycan and lumican for 1 wk postnatally (485).
Interestingly, during this period the structure of the
intramuscular connective tissue changes markedly, thereby the
neonatal structure is less organized than that seen just 2–3 wk later
(672).
The increases in PG expression are paralleled by increases in
myostatin expression and transforming growth factor- (TGF-) and could suggest an interplay between the development
of skeletal muscle and intramuscular
connective tissue (485,
672).
E. Functional Implications of ECM in Tendon and Muscle
It is important to accept that both tendon and intramuscular connective tissue interact closely with the contractile elements of the skeletal muscle to transmit force (521, 562, 566, 567, 573, 610, 675, 691). The dimensions of tendons will influence the ability to stretch, and the ability of the tendon and the intramuscular connective tissue to store and release elastic energy during movement reduces the overall energy need during walking or running (15, 71). Some of the evidence for the functional importance of ECM components stems from studies of mutant knockout models. Given its important role in basal membrane formation, it might be obvious that mice lacking laminin will result in growth retardation and muscle dystrophy. Furthermore, mutations of integrins will also lead to muscle dystrophy and in collagen type VI to myopathy (303, 442). In mice lacking collagen type IX or XI, abnormal collagen fibrils will be found especially in relation to joints (199, 397), while in animals lacking type X collagen chondrodysplasia will develop (666). Furthermore, a defect in types IV, IX, XIII, and type XV collagen will cause myopathy symptomatology (87, 88a, 185, 367). Knockout models for collagen type I, especially when accompanied by mechanical loading, have been difficult to study, in that these animals develop severe osteogenesis imperfecta (126). Finally, somewhat interestingly, in models for proteoglycan defects in the form of a fibromodulin-null mouse, irregular collagen fibrils in tendon structure was observed, whereas no changes were detected in bone or cartilage (615). Mice lacking biglycan and fibromodulin will experience ectopic tendon ossification (27). In line with this, in mice lacking COMP, no clear musculotendinous abnormalities could be found, whereas in humans without COMP, skeletal dysplasias are observed (267). The limitation of these models is the concept of redundance, a phenomenon that is likely to be present also in the ECM, as it can be demonstrated for regulation of circulation and release of hormones in relation to exercise (333, 334).
An important role in linking together the fibrous elements of the ECM whether in muscle or tendon are the proteoglycans (111, 572, 581, 583, 584, 673). Within muscle, it has been demonstrated that PGs in the perimysium are rich in chondroitin and dermatan sulfate. In contrast, those PGs that are present in the endomysium and the basal membrane are dominated by heparan sulfates (484). In addition, decorin has been demonstrated to be present in at least bovine muscle closely associated with chondroitin sulfate (183, 480), and this is dominant in muscles during the early embryonic and postnatal state (644, 645), whereas heparan sulfate is dominant in the late embryonic state. Although several of the ECM substances in addition to collagen have been located in tendon (and muscle), little information on its functional role has been provided. One of the leucine-rich small proteoglycans that envelopes the collagen fibrils is decorin (583). Knockout of decorin suggests the involvement of decorin in the formation of collagen fibrils and to some extent controls the diameter of the fibril and prevents any lateral fusion of collagen fibrils (111, 156). Furthermore, inhibition of decorin results in larger collagen fibrils and increased mechanical properties in healing ligaments (478, 479). The clear role of decorin, or any coordinated effect of either fibromodulin or lumican situated in the same region as decorin, but having different binding sites (268, 616), is not definitively clear (119, 196, 616). More recently, it has been shown in chick embryonic tendon that small leucine-rich PGs like decorin are bound to collagen even before collagen fibril assembly, and this suggests a much earlier involvement of decorin and other PGs than thought so far (245). Even though PGs and glycosaminoglycans are important for tendon function, it has been suggested that neither these nor the collagen fibril size in itself can explain the biomechanical capacities of tendon tissue, therefore suggesting a more complex interplay involving factors and component of tendon tissue yet to be described.
One of the large chondroitin sulfate PGs, aggrecan, is largely upregulated upon compressive loading of the tendon tissue, whereas decorin only responds to tensile loading (548, 549), and is likely to be involved in the preference to synthesize type II collagen in regions of tendons that are subjected to compressive forces (61, 62, 528). Compressed areas of tendon are found to have increased amounts of larger weight PGs (200, 557). This illustrates the differentiated response to tensile and compressive loading, respectively, on collagen and ECM proteoglycans (663, 664).
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IV. REGULATION OF COLLAGEN AND OTHER EXTRACELLULAR MATRIX PROTEIN SYNTHESIS: INFLUENCE OF CHANGES IN MECHANICAL LOADING |
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The major component of the ECM, collagen, is produced in
principal by fibroblasts either on the membrane-bound ribosomes
of the rough endoplasmic reticulum (ER)
or placed within the ECM, respectively. Collagen biosynthesis is
characterized by the presence of
an extensive number of
coand posttranslational modifications of the polypeptide chains, which
contribute to the quality and stability of the
collagen molecule (Fig.
4). Intracellularly, translation of
preprocollagen mRNA occurs in ribosomes and procollagen assembly in
the endoplasmatic reticulum (437,
443,
661).
C-propeptide domains of
polypeptide -chains fold, and trimerization initiates the triple helix
formation in fibrillar collagen types. These events depend on a
well-matched interaction of ER
enzymes like prolyl-4-hydroxylase (P-4-H),
galactosylhydroxy-lysyl-glucosyltransferase (GGT), lysyl hydroxylase,
prolyl-3-hydroxylase, hydroxylysylgalactosyltransferase as well as on
heat shock protein 47, glucose regulating protein 94, and protein
disulfide isomerase (201,
202,
368,
473,
539,
561,
568,
604).
Genetic information for procollagen chain formation is divided into
several exons in the DNA separated by relatively large intron areas
and thus demands extensive processing of RNA prior to that mature mRNA being
available for protein synthesis (661).
Procollagens are transferred from ER to the extracellular space through the Golgi
apparatus and contain NH2-terminal and COOH-terminal
extension peptides at the respective ends of
the collagen molecule (264).
The fact that procollagen is larger that the conventional transport
vesicles necessitates transport within the Golgi apparatus (88).
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After secretion into the extracellular space, the amino-propeptides
are cleaved by specific proteinases and the collagens self-assemble
into fibrils or other supramolecular structures (516)
(Fig.
3). The synthesis of
collagen fibrils occurs first as an intracellular step with
assembling and secretion of
procollagen, followed by an extracellular step converting the procollagen into
collagen and subsequent incorporation into stable cross-linked
collagen fibrils (Fig.
3). The synthesis of type I collagen is used here to
illustrate the fibrillar collagen formation, due to its dominance in
the connective tissue of tendon and muscle, but synthesis
of other collagen types shares many similarities
with that of collagen type I, but is described
further in this review. Following the transcription of
genes coding for the formation of
collagen type I, the pro--chains initially synthesized undergo marked
posttranslational reactions. First, hydroxylation converts residues
to 4-hydroxyproline or 3-hydroxyproline by three different
hydroxylases (473).
Interestingly, the hydroxylases only act on nonhelical substrates and
do not act on collagen or collagen-like peptides that are triple
helical. Newly synthesized collagen polypeptides are glycosylated,
and this process ends before the folding of collagen into a triple helix
structure. Finally, the intracellular processing completes with the
synthesis of both intrachain and interchain
disulfide bonds (170).
This last process is not started before the translation is completed
and probably not before the chains are released from ribosome. The
procollagen is then secreted from the cell, and it is well described
that the rate of
secretion depends on the intracellular processing of
the protein. If folding of the
pro--chains into the triple helical confirmation is prevented,
secretion of the protein is delayed. The three
polypeptide chains form a triple-helical structure. The -chains forming the structure are composed of
repeating amino acid sequences Gly-X-Y, where the glycine residue
enables the three -chains to coil around one another. Proline and 4-hydroxyproline
residues appear frequently at the X- and Y-positions, respectively,
and promote the formation of
intermolecular cross-links. The stability and quality of the
collagen molecule is largely based on the intra- and intermolecular
cross-links. The 4-hydroxyproline formation is catalyzed by P-4-H and
is a unique feature of collagen. Thus its assay is suitable
for evaluating collagen content. Levels of
P-4-H activity generally increase and decrease with the rates
of collagen biosynthesis, and assays of the
enzyme activity have been used for estimating changes in the rate
of collagen biosynthesis in various
experimental and physiological conditions (256–258,
318,
474,
569–571,
619,
620).
The exact location of the processing of procollagen to collagen might however be more complex than that (533). Procollagen N-proteinase (called ADAMTS-2) has recently been cloned (142, 143), and procollagen C-proteinase has been documented to be identical to bone morphometric protein (BMP-1) (327, 398) in which, at least in mouse embryonic tendon, all three protein variants of BMP-1 are expressed (541, 580). Recent experiments in embryonic chicken tendon using pulse-chase followed by sequential extraction have revealed that both intra- and extracellular pools of active procollagen C-proteinase (BMP-1) are present in fibroblasts, whereas the N-proteinase is located in close proximity or within the plasma membrane (91, 111). Conversion of extracellular procollagen to collagen was prevented when procollagen C-proteinase was blocked, whereas on the other hand, collagen fibrils were seen in post-Golgi vesicles and tubules (111). Therefore, despite demonstrated procollagen to collagen conversion being completed extracellularly, some collagen formation may be completed intracellularly and the sequence of C- and N-proteinase in converting procollagen into collagen appears to be more random than so far thought (111).
Fibril segments are prerequisites for fibril formation (496,
497)
and have been shown to increase in length from a few microns to 100 µm (76–78),
and gradually develop increasing diameter (203,
204).
It is likely that fibrils develop into different mass profiles, where some are regular linear ones,
some are very short and spindle-shaped, and some are intermediary
fusing fibers (245,
492).
The collagen molecules are arranged either unipolar or bipolar (245,
312,
313)
and fuse end to end (77,
78).
Most likely as a result of MMP
activity from the fibroblasts, this end-to-end fusion is followed by
increased decorin formation (77,
78)
and removal of collagen type XIV from fibril surfaces
(703).
The fibrillar structure and the tissue's resistance toward loading is
yet to be clarified (135,
231),
but it is known that the cross-link-deficient tendons are less
resistant to loading (680)
and that the elongation of the
tendon depends on molecular gliding within the collagen fibrils (215,
519).
Proteoglycans are important for fibrillogenesis (156,
164,
702),
and when decorin/fibronectrin binding is inhibited, tendon length
is increased (112).
Determination of pyridinoline (Pyr), which represents
important components of cross-links of
mature collagen fibers within the ECM, has shown that the content
of Pyr is especially high in tendon and
ligaments, and interestingly also that the Pyr-to-collagen ratio
appears to be high in tendon and ligament compared with, e.g., bone
(237).
This underlines the likely importance of
cross-links in these structures (339)
and stresses the complexity in studying structure-function
relationship with regard to connective tissue as tendon, ligaments,
and to some extent skeletal muscle (194).
B. Determination of Collagen Turnover in Humans
To determine turnover rates for collagen, radiolabeled amino acids have been introduced (387–389). These allow for labeling of substances such as proline, which are incorporated into the collagen. If infused they will result in an increase in the specific activity of a certain tissue removed from the experimental species at a selected time point. The calculation of turnover rates initially however was based on first-order decay curves that rely on the prerequisite that all collagen molecules are equally likely to be degraded. If, which has been shown, the collagen pool is subdivided into a more fast and a more slow exchanging pool, this method likely gave a pessimistic picture of the capability to turnover collagen (387). More recently, the use of techniques "flooding" the precursor pool to reduce or eliminate reutilization and using infusion over a relatively short time has been performed successfully (44).
Microdialysis of both muscle and connective tissue has been performed with the intention to mimic the function of a capillary blood vessel by perfusing a thin dialysis tube with a physiological fluid implanted into the tissue (384–386). This allows for collection of extracellular fluid both in animals and in humans in vivo, both with the organism in a basal state as well as during conditions where physiological perturbations are performed, whether these are chemical or mechanical. The collection of dialysate allows for calculation of interstitial concentration of unbound substances that are able to cross the membrane of the catheter, provided the technique is supplemented by calibration methods that allow for quantitation of this. Using microdialysis fibers along the peritendon also provides the possibility to study during exercise. For several metabolic parameters it has been shown that determinations peritendinously reflect the changes that occur intratendinously (379).
C. Responses to Increased Loading: Acute and Chronic Exercise
Muscular and tendinous collagen and the connective tissue network are known to respond to altered levels of physical activity (347–350, 507, 612, 613, 623, 658, 682, 688, 706). The specific activities and content of collagen components are known to be greater in the antigravity soleus muscle than in the dorsiflexor tibialis anterior, which is not tonically active (349, 569). Furthermore, ECM in skeletal muscle is known to respond to increased loading caused by endurance training (348, 349, 619, 711), acute exercise (474), or experimental compensatory hypertrophy (676) by increased collagen expression, synthesis, and collagen accumulation in the muscle. Strenuous exercise, especially acute weight-bearing exercise that contains eccentric components, is known to cause muscle damage (33). Upregulation of collagen synthesis may be a part of the repair process but may also occur without any evidence of muscle damage (256). Acceleration of collagen biosynthesis after exercise may thus reflect both physiological adaptation and repair of damage.
It has been found in mice that acute exercise increases activities of enzymes of collagen turnover 48 h after exercise. Enzymes responsible for collagen synthesis were increased, and the most in muscles dominated by red rather than white muscle fibers (474, 650, 651). This fits with the demonstration of higher content of collagen, as measured by amount of hydroxyproline, in red versus white muscle fiber dominated muscles, and with a high recruitment of red fiber muscles during the specific exercise protocol (349). Interestingly, the changes in collagen enzyme activities were accompanied by a rise in both hydroxyproline and collagen content of the exercised muscle, which persisted up to 3 wk after exercise (474, 650, 651). Later studies have demonstrated that expression of types I and III collagen was increased at the mRNA level within 1 day (257, 345) and that of type IV collagen as early as 6 h after acute exercise (344).
Extracellular conversion of procollagen to collagen requires at least two enzymes: a procollagen aminoprotease that removes the aminopeptides and a procollagen carboxyprotease that removes the carboxy propeptides. The cleavage of the carboxypeptide allows for indirect determination of collagen type I formation. Development of assays for such markers of type I collagen synthesis [the COOH-terminal propeptide of type I collagen (PICP)] and degradation [the COOH-terminal telopeptide region of type I collagen (ICTP)] has made it possible to study the effect of exercise on collagen type I turnover (99). When determined in circulating blood, these markers have been shown to be relatively insensitive to a single bout of exercise, whereas prolonged exercise or weeks of training were shown to result in increased type I collagen turnover and net formation (383). However, as bone is the main overall contributor of procollagen markers for collagen type I turnover in the blood, and as serum levels of PICP and ICTP do not allow for detection of the location of the specific type of region or tissue in which changes in turnover are taking place, from these studies it cannot be concluded whether changes in collagen turnover of tendon-related tissue or intramuscular connective tissue occur. The use of the microdialysis technique has recently been applied to the peritendinous space of the Achilles tendon in runners before, immediately after, and 72 h after 36 km of running (386) (Fig. 5). With this technique it was demonstrated that acute exercise induces changes in metabolic and inflammatory activity of the peritendinous region (386). In addition, acute exercise caused increased formation of type I collagen in the recovery process, suggesting that acute physical loading leads to adaptations in non-bone-related collagen in humans.
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Furthermore, when type I collagen synthesis and degradation in connective tissue of the Achilles peritendinous space was studied before and after 4 and 11 wk of intense physical training, an adaptive response of the collagen type I metabolism of the peritendinous tissue around human Achilles tendon was found in response to physical training (382) (Fig. 5). The increase in interstitial concentrations of PICP rose within 4 wk of training and remained thereafter elevated for the entire training period, indicating that collagen type I synthesis was chronically elevated in response to training. As blood values for PICP did not change significantly over the training period, it is reasonable to suspect that the increased collagen type I formation occurs locally in non-bone tendon connective tissue rather than reflecting a general rise in formation of collagen type I throughout the body (382). Also, tissue ICTP concentrations rose in response to training, but this rise was transient, and interstitial levels of ICTP returned to basal levels with more prolonged training. Taken together, the findings indicate that the initial response to training is an increase in turnover of collagen I, and that this is followed by a predomination of anabolic processes resulting in an increased net synthesis of collagen type I in non-bone connective tissue such as tendons (382). The pattern of stimulation of both synthesis and degradation with the anabolic process dominating in response to exercise in tendon-related connective tissue is a pattern that is in accordance with events occurring with muscle proteins in response to loading (540). As individuals in the training study (382) were training on a daily basis, it can be difficult to differentiate effects of each bout of acute exercise from the chronic training adaptation. Previous acute bouts of exercise will influence the outcome of each subsequent one. This probably explains why highly trained runners (training up to 12 h/wk) in one study had high basal levels of interstitial levels of PICP (386). Thus it cannot be excluded that the effect on collagen metabolism found during a program with daily training simply reflects an effect on collagen formation from the last training bout, rather than chronic effect of training.
On the basis of these findings it can be concluded that both an increased collagen turnover is observed in response to training and that with prolonged training a net synthesis of collagen type I is to be expected. Whether a net synthesis of collagen type I is transformed into morphologically detectable increases in tendon size is far from clear. However, in accordance with this view, it has been demonstated in animal models that training results in enlargement of tendon diameter (74, 605, 689). Furthermore, recent cross-sectional observations in trained runners versus sedentary humans have shown that magnetic resonance imaging (MRI)-determined Achilles tendon cross-sectional area was enlarged in trained individuals compared with untrained controls (553). It can also be speculated that training initially results in an increased turnover of collagen type I to allow for reorganization of the tissue and that more prolonged training results in a net increase in tendon tissue and probably alterations in tissue strength.
No clear dose-response relationship between type and amount of training and adaptive responses of collagen formation exists, but in equine tendon it has interestingly been found that in tendon subjected to low-level repetitive stress (extensor tendon) the collagen level is higher that in flexor tendons subjected to high stress (497). This indicates that intensity and loading pattern including recovery periods between training bouts likely play an important role in adaptation of ECM. Stretch-induced hypertrophy of chicken skeletal muscle has been shown to increase muscle collagen turnover using tracer methods (389), which is in accordance with the present findings on humans in which collagen synthesis increased markedly at the beginning of training. From studies by Laurent and co-workers (387–389), it was concluded that a large amount of newly synthesized collagen was wasted, resulting in disproportionate high collagen turnover rate compared with the magnitude of net synthesis of collagen. Likewise, in human muscle, type IV collagen degradation, as indicated by an increased MMP-2, increased over a period of 1 year with electrical stimulation of spinal cord injured individuals, without any detectable change in type IV content, indicating an increased collagen turnover rate with no or very little net synthesis (342). Measuring the racemization and isomerization of its C-telopeptide (CTx) has been used to determine collagen type I turnover. As these processes are coupled to protein turnover it is likely that they reflect an index of collagen turnover (237). With this method it has been shown in human cadaver tissue that tissues like tendon and ligament have a turnover rate comparable to that in bone, and that the turnover of collagen type I is in fact quite pronounced in skeletal muscle (237). These data agree with studies using radiolabeled proline/hydroxyproline in animals showing a relative turnover (3%) of collagen per day in skeletal muscle (389).
The present studies in humans and animals support the idea of a simultaneous activation of both formation and degradation in collagen of both muscle and tendon tissue in response to loading. Interestingly, for type I collagen in tendon, timewise this is followed by a more pronounced imbalance in favor of formation and resulting in a net collagen synthesis, whereas in muscle, type IV collagen does not seem to reveal any net synthesis. In addition to an increased turnover of collagen, indirect evidence for loading increased both synthesis and degradation rates has been demonstrated also for non-collagen components of the ECM (53, 537, 549).
An important step in collagen type I formation is the enzymatic
regulation by procollagen C-proteinase (PCP) of the
cleavage of PICP and PINP of
procollagen to form insoluble collagen (311).
It has been demonstrated that mechanical load can enhance the
expression of the PCP gene, but not of the
procollagen C-proteinase enhancer protein (PCPE) in dermal
fibroblasts (498).
In addition to this, it was shown that both the synthesis and the
processing of procollagen were enhanced by loading in vitro.
This effect was demonstrated to be specific as non-collagen protein
synthesis in that study was not elevated (498).
Furthermore, an enhanced processing of
procollagen to insoluble collagen was found, as evident by a larger
increase in the actual amount of
insoluble collagen produced compared with the increase in
procollagen synthesis. Interestingly, in that study it was
demonstrated that the above-described changes only occurred if cells
were in tissue cultures containing TGF- or serum (498),
indicating that mechanical loading by itself is not able to cause
changes unless certain growth factors were present.
D. Immobilization and Collagen Turnover
In contrast to physical loading, immobilization of rat hindlimb leads to a decrease in the enzyme activities of collagen biosynthesis in both skeletal muscle and tendon (569, 570), which suggests that the biosynthesis of the collagen network decreases as a result of reduced muscular and tendinous activity (181). The rate of the total collagen synthesis depends mostly on the overall protein balance of the tissue, but it seems to be positively affected by stretch in both muscle and tendon (569, 570). Changes in the total collagen content of muscle, measured as hydroxyproline content, are usually small or absent during immobilization lasting for a few weeks, which is probably due to the turnover rate of collagen (257). Collagen expression during immobilization has been shown to be at least partially downregulated at the pretranslational level (11). The mRNAs for type I and III collagens were already decreased after 3 days of immobilization, whereas stretch seemed to counteract this decrease (11). The content of type IV collagen was also reduced as a result of immobilization (12).
In other studies of rats, amounts of collagen in skeletal muscle were studied in response to immobilization at increasing muscle length to cause either atrophy or hypertrophy (569). Whereas P-4-H decreased and proteolytic enzyme activity increased in shortened muscle, no increase in P-4-H or GGT could be detected in lengthened muscle (569). Electrical stimulation could, at least in some muscles, counteract the immobilization-induced drop in muscle mass together with the decrease in content of hydroxyproline and collagen-related enzyme activities (571). To some surprise, it was found that denervation of muscles in rats resulted in an elevation of hydroxyproline content and in P-4-H and GGT activities of muscle (571). Although non-collagen proteins of the atrophying muscle were degraded at a high rate during denervation, this could not solely explain the rise in collagen content and enzyme activity with denervation. In tendons of immobilized and denervated muscle, activities of collagen-synthesizing enzymes fell during immobilization in the shortened position but were unaffected when immobilized in the lengthened position (571). This indicated that the regulation of collagen synthesis to varying mechanical loading possessed similarities in skeletal muscle and tendon tissue. Further studies of remobilization of skeletal muscle after 1 wk of immobilization in rats showed that activities of P-4-H and GGT as well as concentrations of hydroxyproline (HP) rose within days, whereas remobilization exercise did not cause any rise in tendon P-4-H or GGT (318). This could imply that although activity can activate collagen synthesis in both tendon and skeletal muscle, a higher activity is needed to stimulate tendon than muscle.
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V. DEGRADATION OF CONNECTIVE TISSUE IN TENDON AND SKELETAL MUSCLE: EFFECTS OF CHANGES IN MECHANICAL LOADING |
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Collagen degradation is initiated extracellularly by MMPs (or matrixins), which are presented in tissues mostly as latent proMMPs (38, 49, 475). There is evidence to support that MMPs [and tissue inhibitors of matrix metalloproteinases (TIMPs)] are not involved to a major degree in the intracellular lysosomal phagocytosis, but function extracellularly (192). The collagen degradation processes are well described in situations with rapid remodeling, e.g., inflammation or tissue damage (140, 295, 436), but its role during normal physiological stimulation to increase tissue turnover like after mechanical loading is not known (192). Whereas collagenases (MMP-1 and MMP-8) traditionally are thought primarily to initiate degradation of type I and III collagen and thus should be most relevant for tendon, gelatinases (MMP-2 and MMP-9) mainly break down nonfibrillar type IV collagen and other compounds of the ECM. Although some preference for the different MMPs with regard to collagen types exists, the specificity of certain MMPs toward collagen types may be less than thought so far. As an example, MMP-2 can also degrade type I collagen (13), most likely in a two-step fashion (500), and the whole role of MMPs on tissue matrix metabolism seems far more complex (475). MMPs are produced from endotenon fibroblasts and intramuscular matrix fibroblasts, although the secretion is somewhat lower than that of synovial cells (526).
Immobilization leads to an increase in MMP expression at both
preand posttranlational levels, suggesting accelerated collagen
breakdown, which can be partially prevented by stretching procedures.
The regulation of MMPs in relation to exercise remains to be
fully understood, but it is known that specific integrins (21) are regulators of the
MMP-1 gene expression in fibroblasts cultured in
contracting-retracting collagen gel (475).
Furthermore, it is known that MMP-1 expression can be modulated by
growth factors, inflammatory cytokines, and cytoskeleton-disrupting
drugs like cytochalasin D. However, the role
of contraction and stress relaxation
has not been evaluated. In a retracting collagen gel, MMP-1 is
expressed as a result of the tension on the tissue (369)
and mediated through 21-integrins (545).
Increased fluid flow has in vitro been shown to increase the
expression of both MMP-1 and MMP-3, together with an
activation of interleukin (IL)-1 and cyclooxygenase (COX)-2 genes (29,
32).
Mechanical stress relaxation stimulated MMP-1 gene expression has
more recently been shown to depend on de novo protein synthesis,
although it occurs independently of the
activation of an IL-1 autocrine feedback loop (370,
371).
Mechanical stretching is known to elicit an increase in MMP gene
expression (29,
32)
without any obligatory rise in collagen type I expression or
indication of changes in inflammatory meditors in vitro.
However, the IL-1 was able to induce marked increases in MMP expression, and
a synergistic effect of IL-1 and stretching was observed (29,
32).
This fits with findings on human tendon tissue where IL-1 and oncostatin caused an increased production of
MMP-1 (117).
In lung fibroblasts, stretching is found to increase activation
of MMP-9 directly in the absence of any
inflammatory mediators (617).
Interestingly, shear stress elicited by fluid pressure on rabbit
tendon fibroblasts elicits expression and release of
MMP-1 and -3, in the absence of any
change in intracellular calcium concentration (29,
32).
MMP-2 and -9 are known to be overexpressed and present in higher
amounts in patients with inflammatory myopathies (329),
which may increase ECM degradation and thus facilitate lymphocyte
adhesion (132).
Furthermore, the attachment of
type I collagen to cultured fibroblasts upregulated MMP-2 and MMP-9
production, an effect that was blocked by dexamethasone (547).
Finally, MMP-2 is colocalized with integrins placed in vessels (102),
and MMP-mediated proteolysis of
capillary basement membrane proteins is important for physiological
angiogenesis responses to chronic loading of
skeletal muscle, and increased production of
MMP-2 and MT1-MMP is crucial for new capillary formation during
mechanical stimulus (251,
252,
641).
Taken together, several of the
exercise- and training-related adaptations are coupled to processes
that are found to involve MMP responses (409).
Recently, it has been demonstrated that acute exercise resulted in
elevated interstitial amounts of
MMP-2 and MMP-9 in human peritendinous tissue (346),
which supports the view that MMPs (and their inhibitors) play a
role in ECM adaptation to exercise in
tendon tissue.
TIMPs inhibit MMP activities (198). Of the four TIMPs so far identified, TIMP-1 and TIMP-2 are capable of inhibiting activities of all MMPs with a preference for inhibiting MMP-2 and MMP-9, respectively. Pro-MMP-2 is upregulated both at the pre- and posttranslational level after a single bout of exercise, suggesting an increase in the collagen degradation (343, 345). In line with this, stretch-relaxation on dermal fibroblasts results in increased activity of MMP-2 through integrin-mediated pathways (369, 372). As mentioned previously, increased MMP activity and thus enhanced degradation of collagen often parallels the stimulated activation of collagen synthesis (345). Interestingly, TIMPs are often activated together with MMPs in response to physical activity (345), indicating simultaneous stimulation and inhibition of degradation. Rather than considering this as a competitive action, it is likely that MMP activity precedes TIMP activity, and thus TIMP serves as regulators of degradation termination to ensure a limited amount of degradation. To support further that an integrated response of MMPs and TIMPs exist, TIMP-2 has been shown to be important for an activation of pro-MMP-2 in vivo (670). TIMP-1 has been found to be correlated with ICTP in patients with cancer, indicating activity in and control of collagen degrading pathways (701).
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VI. STRUCTURE OF EXTRACELLULAR MATRIX IN TENDON AND MUSCLE: RELATION TO MECHANICAL AND VISCOELASTIC PROPERTIES |
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Tendons vary in their ability to stretch, from 1–2% of lengthening of the animal extensor carpi radialis, to 3–4% of lengthening of the flexor carpi ulnaris tendon, and up to 16% of elongation of the rabbit Achilles tendon (403). Human cadaver data imply that the maximal elongation of human tendon is up to 5–6% when passively stretched (413). In association with this, the aponeurosis of the adjacent muscle generally displays a larger mechanical excursion compared with free tendon under passive stretch (10–12%) (403). Although indicative of the viscoelastic potential of the tissue when challenged to passive loading, these findings do not take into account the in vivo characteristics during muscular activity. Some studies have shown that the free tendon and aponeurosis of the cat triceps surae have comparable mechanical properties during isometric contraction (585, 629). In contrast, the stiffness of the aponeurosis is found to be less than that of free tendon during contraction, whereas others have found the reverse, namely, that tendon strain was 2% while aponeurosis strain was 8% during passive loading (403) (Fig. 6). Such findings do not, however, take into account that during muscle contraction in vivo in humans it is likely that the contractile apparatus of the muscle will limit the excursions of the aponeurosis. Attempts to separate aponeurosis movement and free tendon movement in vivo have shown varying results (296, 422). Some studies found no difference between Achilles tendon and the adjacent aponeurosis (225, 226, 355, 356). However, in those studies the Achilles tendon was defined as the free tendon plus the soleus aponeurosis. In a study on tibialis anterior free tendon, this was only strained 2%, whereas the aponeurosis was strained up to 7% during submaximal isometric contraction (422). Somewhat in contrast, it has been shown for the human Achilles tendon that the strain of free tendon was six- to sevenfold greater than that of the aponeurosis during intense isometric calf muscle activation (427) (Fig. 6). The difference between studies of viscoelastic properties of the free tendon and skeletal muscle aponeurosis can both be accounted for by measurement limitation, such as accounting for antagonist activity, joint rotation, and the fact that the methodology is limited in its ability to observe three-dimensional phenomenon (85, 425). The pronounced difference between the strain in free tendon and the aponeurosis in the human Achilles region suggests that their functional role during force transmission differs. It is on this background suggested that free tendon permits for storage and release of energy, while the aponeurosis ensures effective transmission of contractile forces.
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B. Repetitive Loading and Tendon Properties
Several in vitro systems for determination of cellular responses to mechanical tensile stress are available. One way to study mechanical loading of fibroblasts is to seed cells upon a flexible substrate that allows for easy control of strain, whereas actual forces put upon the individual cell cannot be accurately quantified (221, 418). Another method to grow fibroblasts is in a native three-dimensional collagen matrix in which they attach and pull on collagen fibrils. This latter method allows for determination of tensile force developed, whereas strain is more poorly defined (221). It is clear that fibroblast cultures subjected to biaxial mechanical loading often result in uneven strain and loading across the plate, and this model thus only obtains results that represents a mean of the different ranges of movements (498). The in vitro systems evidently have major advantages with regard to localized measurements and the possibilities for intervention; however, it still represents a simplified system that does not always encompass all the growth factors that are needed in the transformation phase from mechanical loading to synthesis of ECM proteins like collagen.
Tendons respond to mechanical loading, and animal studies have provided some evidence that endurance training will influence their morphology and mechanical properties (106, 190, 355, 652, 687, 689). In rabbits, it has been shown that the load-deformation curve of the Achilles tendon was unaffected by 40 wk of training, which implies that no structural properties were influenced by the training (652). However, the same group also showed with similar training that the posterior tibialis tendon displayed a load-deformation curve that was altered without any detectable change in tendon volume or mass, which suggests that the mechanical loading resulted in qualitative rather that quantitative changes (653). Somewhat in contrast, 12 wk of training in swines increased both load-deformation and stress-strain properties of digital extensor tendons, together with an increase in the cross-sectional area (CSA) and total collagen content of the tendon (687, 689). This supports the view that both structural as well as mechanical properties are improved with training. Interestingly, in a similar model, this was shown to be the case only after 12 mo, but not after 3 mo of training, and the shorter training period in fact reduced the tendon size (687). In support of this pivotal adaptation of tendon morphology, training studies on horses revealed that 5 mo of training did not influence the CSA of the Achilles tendon, whereas 18 mo of training increased it by 14% (74, 502–504). Finally, in rats the CSA of the Achilles tendon reduced after 1 mo, but increased after 4 mo of training (505). In both the latter studies a quasi dose-response relationship was obtained in that low-intensity training did not change the tendon morphology, but intense training did so. In two additional studies, training did not alter the dry weight of the patellar tendon of rats (638) or of chicken Achilles tendon (153). A study in turkeys showed that despite only a minimal change in tendon CSA area after training, the mechanical properties changed and an increased tendon stiffness was found (106). This would theoretically yield a larger amount of stored energy in the tendon and thus enhance locomotion economy. This would, however, require a larger force on the tendon, which is unlikely at the same absolute running speed, and it was therefore suggested that the altered properties could contribute to a larger tendon resistance towards material fatigue and subsequent damage.
Somewhat in contrast to other studies, running in mice resulted in unchanged tensile strength of patellar tendon (330), but it has to be acknowledged that the mice were not adult, as has been the case in other studies (652, 687, 689). Finally, rat Achilles tendon was found to increase tensile strength and stiffness after 30 days of training (655). In addition to the classical stress-strain curves, attempts have been performed to address cumulative damage and fatigue development in tendon tissue that is repeatedly loaded with forces that are below the ultimate tensile stress. It was found that time-to-rupture among tendons was similar when tendons were subjected to a load that corresponded to the maximal voluntary contraction of their corresponding muscle (324–326). This would mean that all tendons appear similarly prone to fatigue ruture, and it is suggested that any CSA of a tendon is coupled to the achievement of a certain stiffness.
Only one study has tried to compare strength and endurance training, and in rats they found that 38 wk of training was associated with an age-related decline in ultimate load-to-failure that was counteracted by swimming training, but no effect was seen in response to strength training (595). It could be argued that the strength protocol was limited in that study and that a further major drawback was the lack of any determination of tendon area or volume, which precluded the evaluation of any quantitative versus qualitative effect of training. Only few studies have addressed the effect of training on intratendinous structures (159), and although one very early study did not demonstrate any intratendinous fibril increase as a result of training in rats (292), a subsequent study demonstrated increased fibril diameter after training (453, 454). In the horse, an 18-mo training program did not result in any significant change in collagen fibril diameter of the deep flexor tendon of the horse (502, 504). As horses have been shown to increase the CSA of the tendon in response to such prolonged training (74), it indicates that the number of collagen fibrils was increased. This was also found in one study after 10 wk of training (454), and furthermore, one study demonstrated more densely packed and aligned fibrils as a result of training (655).
It has been shown with ultrasonography in humans that the compliance of the muscle vastus lateralis aponeurosis-tendon complex was lower in long-distance runners compared with that in untrained subjects (355). It was stated that this allowed the complex to store energy and reuse it more efficiently in runners compared with sedentary counterparts. Somewhat in contrast to this, no difference in compliance was found between sprinters and control individuals with regard to both the quadriceps and triceps surae tendon-aponeurosis complex (356). This is interesting, since sprinters are a group of athletes that would need capacity to reuse elastic energy. The same authors found, however, that 8 wk of isometric strength training, but not 4 wk, did increase the stiffness of muscle vastus lateralis in humans, indicating an effect of training duration on tendon-aponeurosis properties (354). However, it has to be remembered that the ultrasonography method used in those studies cannot clearly separate the tendon properties per se from those of the combined tendon-aponeurosis complex.
The highest tolerable tensile load of a
tendon is known to depend on its CSA in relation to integrated
fascicle CSA of the adjacent muscle, and this
relationship varies between tendons in different species but also
between tendons in a given individual (63,
64,
325,
326,
512).
In this respect it is interesting that a cross-sectional study found
that the CSA of long distance runners was 20–30%
larger than untrained controls, while the load-deformation curve
of the triceps surae aponeurosis-tendon complex
did not differ (553).
When the ratio between muscle and tendon area (multiplied by 0.3 MPa
as a chosen number for maximum isometric stress, Ref. 325)
was related to a certain load, differences in fatigue resistance to
failure were seen between different tendons (326)
(Fig.
7). Interestingly, if the individual tendons were subjected to
individual loading dependent on the capacity of the
relevant adjacent muscle, the time to rupture was similar between
tendons (326).
This implies that fatigue and resistance to rupture are coupled to
the tendon-loading pattern, which was shown when high-stress and
low-stress tendons in sheep develop their functional capacity matched
the working demands of the tendon (510).
If we combine this with the data obtained in humans of
varying training degree, the larger CSA of the
trained tendon results in a lower stress on the tendon during maximal
isometric force in trained compared with untrained individuals (553),
and thus provides a potentially more injury-resistant tendon
(Fig.
8). The safety factor (fracture stress set at 100 MPa divided by
the stress during intense activity) has been found to be 8 in general for most tendons (325).
It was shown in humans in vivo that the safety factor was only 2–3
during maximal isometric contraction (431).
Therefore, tendon hypertrophy would seem helpful in counteracting
overload and prevention of
ruptures. These data fit with previous observations on old
individuals where there is a tendency toward larger CSA of the
well-trained individuals (314,
426).
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Sonographic evaluation of the Achilles tendon has shown that the CSA of the tendon is larger in individuals with an active history of physical training than in sedentary counterparts (700). Most likely, increase in tendon CSA in humans requires very prolonged anamnesis of training, as 6 mo of recreational running training in previously untrained individuals was not sufficient to increase the CSA of the Achilles tendon (428).
In muscle, the passive movement of the perimysium is viscoelastic but does not involve any major reorientation movements of collagen fibers in a proteoglycan matrix (519). Rather, the viscoelastic pattern to stretch depends on relaxation processes within the collagen fibers themselves or at the fiber-matrix interphase. In tendon it has been shown that the molecular packing of collagen is extended upon stretch in the rat tail (215, 460). This could be due to molecular straightening and gliding of collagen molecules. However, the strain within collagen fibrils is always smaller than stretching of the entire structure (i.e., the tendon), which implies that additional gliding occurs at the interfibrillar level. That other structures than the collagen fibrils themselves are involved in tension resistance and subject to injury is supported by the demonstration that fibrils do not run through the entire length of a tendon but are largely dependent on cross-linking (630, 632). In line with this, it has been shown that shorter specimen samples of tendons are more resistive to rupture when determining time to failure towards a given test stress, indicating that strengthening structures are added and increasingly important when longer tendons are studied (669). The cross-linking between parallel collagen molecules includes both aldimine and ketoimine, of which the former dominates during development of tendon (47, 48). Later on, other cross-links bind collagen molecules together including pyridinoline, and with aging a nonspecific cross-linking through glycation comes into play (54). Only a few studies have looked at the importance of these cross-links, but it has been demonstrated that chemical preparation of tendon to reduce numbers of almidine cross-links was found to increase tendon strength and diminish stress relaxation, implying that these links are involved in the stress relaxation (160).
With aging of connective tissue or in diseased states with elevated glucose levels (e.g., diabetes) the nonspecific cross-linking mediated by condensation of a reducing sugar with an amino group and result in accumulation of advanced glycation end products (AGEs), in tendon tissue (297, 535, 587). It has been shown that glycated tendons could withstand more load and tensile stress than nonglycated tendons (535), but the tissue gets stiffer (648). In chondrocytes it has been demonstrated that not only is a relatively low turnover of collagen in the tissue a prerequisite for the formation of AGEs (648), but that the presence of AGEs feedback to reduce both collagen synthesis and MMP initiated collagen degradation (162). When biochemical markers (pentosidine and fructosamine) of AGEs are correlated to microscopic determination of collagen fibrils in the rat tail tendon, it can be shown that high amounts of AGEs are coupled to tight assemblence and fusion of fibrils, thus displaying larger collagen fibril diameters than in the healthy (293, 489). Likewise, the morphological and biochemical picture of tendons in which AGEs were induced by glucose incubation were strikingly similar to tendons from diabetic animals (361, 489). The accumulation of AGEs with aging thus indicates a stiffer and more load-resistant tendon and intramuscular ECM structure, but on the other hand reduces the ability to adapt to altered loading, as the turnover rate of collagen is markedly reduced (360). In addition to this, it has been shown that AGEs upregulate connective tissue growth factor (CTGF) in fibroblasts that thus favor the formation of fibrosis over time in elderly individuals and patients with diabetes (636).
It is well described in animals that the average fibril diameter increases during development (118, 496, 497) but declines with aging (476) and disuse (477). The effect of training on this is variable (453, 501–504). Somewhat in contrast to animal data, the overall CSA of the Achilles tendon is larger in elderly compared with younger individuals (426). The phenomenon may reflect a compensatory increase in ECM to lower stress on the tendon due to age-related decline in tendon quality and thus in maximal tolerable load (426). This is interesting, since there are observations of reduced expression and synthesis of collagen in aging fibroblasts (47, 124, 239). In addition, covalent intramolecular cross-links are known to increase the elastic modulus, and reduce strain to failure, but do not influence rupture stress (47, 168, 624). This phenomenon is more pronounced in high-load bearing flexor tendons compared with low-stress extensor tendons (47, 73). Interestingly, cross-linking and stiffness are increased with aging, whereas endurance training counteracts these phenomena (244). The same counteractive effect is found in elderly with low-load resistance training (357), but not found with very intense strength training (538). The fact that in association with increased tendon stiffness with aging, bone mineral density is decreasing, which might explain why older individuals are more prone to get avulsions of the bone rather that tendon ruptures compared with younger counterparts (692).
D. Integrated Muscle-Tendon ECM Properties
In vivo determination of viscoelastic behavior of a stretched muscle-tendon unit (423, 424). In the absence of any detectable electromyelogram (EMG) activity in the passively stretched muscles, and during dynamic stretching a curvilinear increase in resistance towards stretch was found, whereas a nonlinear stress relaxation response declining the resistance by 25–35% was demonstrated over the 45 s of static loading phase (433). This effect has no influence on subsequent stretch procedures (424, 435, 443). Furthermore, when a series of stretching procedures was carried out, it was found that this acutely lowered the stiffness and storage of passive energy during dynamic stretch, but that the effect vanished 1 h after stopping the stretching regimen (432–434). This supports the notion that passive stretching of muscle-tendon has no chronic effect on viscoelastic properties of the tissue. If this is the case, chronic stretching of muscle tendon unit in association with physical training should not cause any altered passive properties of the muscle-tendon unit, and thus challenge common clinical belief (227). In an investigation where subjects performed repeated stretching exercises twice daily for 3 wk, no change in passive properties of the muscle-tendon unit was found. It was demonstrated that the maximal flexibility was improved by the training, but this was achieved at the expense of a high resistance to stretch (434). At present it is unknown what mechanism lies behind this phenomenon. Evidently, the passive properties interplay with active muscle contraction in a very complex way. In isolated animal muscle-aponeurosis-tendon preparations it has been shown that rapid elongation of the passive tendon structure takes place upon muscle stimulation and shortening (404). This potentially creates a scenario where muscle fibers shorten at a high velocity and lower force development in the initial phase of contraction. When the passive structure such as tendon subsequently reaches its point of increased resistance towards stretch, the shortening velocity of the muscle will markedly decrease, and thus allow for a higher force output from muscle (404). Perhaps, depending on tissue qualities, tendon and/or muscle will be more fragile toward rupture at high loading, and that muscle rupture can occur at a late time point in the contraction. In addition, surgical transfer of tendon in human arm has been demonstrated to influence functional muscle-tendon performance dependent on the length at which the muscle-tendon complex is surgically inserted (219). Thus an attempt to overstretch the tendonmuscle when inserting will cause the sarcomere length of the muscle to be at a suboptimal length with regard to force development (219).
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VII. LOADING AND OVERLOADING OF CONNECTIVE TISSUE STRUCTURES IN TENDON AND SKELETAL MUSCLE: ROLE OF GROWTH FACTORS |
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, IL-1, IL-6, and IL-8, insulin-like growth factor I
(IGF-I), fibroblast growth factors (FGF), NO, prostaglandins,
vascular endothelial growth factor (VEGF), and platelet-derived
growth factor (PDGF), all have positive effects on fibroblast
activation in vitro, when fibroblasts from different tissues
are used (14,
25,
68,
234,
259,
262,
518,
633).
However, in most studies no isolated attempt has been undertaken to
compare this effect with any separate or simultanous role
of mechanical loading upon the release
or synthesis of such growth factors (536).
In one study the importance of
PDGF and mechanical strain was shown to be related to PDGF (51).
Insulin-like growth factors (IGF-I and -II) are known to influence
fibroblast in vitro (7,
8,
10,
51,
121,
576)
and are likely to represent an important growth factor, but their
exact role for collagen synthesis of human tendon and muscle remains to
be elucidated. Furthermore, it has been shown that mechanical loading
induces increased secretion of
TGF-, PDGF and basic FGF (bFGF) as well as expression
of growth factors from human tendon fibroblasts
(598).
Further mechanically loaded fibroblasts result in an increased
gene expression for collagen and ECM components (53).
The effect of specific growth factors or serum components in
combination with mechanical loading on procollagen synthesis and
procollagen propeptide gene regulation indicate a synergy between
signaling pathways with regard to procollagen gene expression and
processing, similar to what has been documented in cardiac
fibroblasts (110).
Interestingly, in a study where tendon grafts used for reconstructive
surgery after ruptured anterior cruciate ligament were analyzed
for content of growth factors, only bFGF was present in
the control situation (359).
Following reconstruction, increased levels of
immunostaining of TGF- and PDGF were noted over the following weeks and were back
to initial levels after 12 wk (359).
Only a few of the most important growth factors for ECM in tendon and muscle will be dealt with in the following section. This should, however, not distract from the fact that others also play major roles and that even more factors are suggested but remain unexplored. As an example, the role of NO and prostaglandins seems clear with regard to mediating mechanical signaling to the process of bone formation in the skeleton (134), and it has been shown that this involves fluid-induced wall-shear stress (599) and that osteogenesis acts through COX-2-dependent pathways (708). This fits with findings of increased release of prostaglandins from human tendon fibroblasts that have been mechanically stimulated in vitro (24) and from human tendon tissue in vivo during mechanical loading (385), and suggests a role for prostaglandins in the adaptation of ECM in tendon tissue (543).
TGF- with its three mammalian isoforms
are known to function as modulators of ECM
proteins and to cause induction of
both collagen gene activation and protein formation via Smad
proteins (28,
123,
158,
289,
304,
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