muscle injury, regeneration, and repair

Address all correspondence and request for reprints to:Peter A. Huijbregts13826 South Meyers Road, #2034Oregon City, OR [email protected]

Muscle Injury, Regeneration, and Repair

Abstract: This article reviews relevant muscular anatomy and describes the metabolic, tempera-ture, and mechanical hypotheses as possible mechanisms of muscle injury. It describes the fourstages of muscle injury, regeneration, and repair: Ca2+-overload, autolysis, phagocytosis, and re-generation/repair. The article concludes with some likely clinical implications for prevention andtreatment of muscle injury.

Key Words: Muscle, Injury, Regeneration, Repair

Peter A. Huijbregts, PT, OCS, FAAOMPT

A plethora of information is available in physical therapyliterature on injury, regeneration, and repair of the

connective tissues. This is understandable: after all, thedifferent types of connective tissue (CT) make up a largepart of the body. Knowledge of CT physiology and patho-physiology forms the basis for prevention and treatmentof many injuries. In contrast, information on muscle in-jury, regeneration, and repair is not as readily availableto physical therapists, yet muscle injury is common. Itcan be the result of mechanical forces; excessive tensionmay be generated during a passive stretch or a contrac-tion, especially a lengthening or eccentric contraction1-

4. Excessive mechanical force is also the reason for muscleinjury due to contusions2, 4-10 and lacerations9, 10. Muscleinjury can result from thermal stress, such as extremeheat or cold1, 6,7. Myotoxic agents can cause muscle in-jury. The local anesthetics marcaine6 and lidocaine incombination with epinephrine10 have been shown to causemuscle fiber necrosis. Excessive doses of corticosteroids10

and certain snake and bee venoms are also myotoxic1.Prolonged ischaemia also disrupts muscle; clinically thismay be seen in compartment syndromes but also afterthe tourniquet application commonly used to create abloodless field for surgical procedures4, 6,9-11. The goal of this article is to increase the therapist’sunderstanding of muscle injury, regeneration, and re-pair. An increased understanding of the process of muscleinjury and subsequent regeneration or repair can pro-vide us with a theoretical basis for more appropriateprevention and treatment of muscle injuries. In this articlerelevant anatomy is reviewed. This will better allow fora discussion of possible causative mechanisms and thestages of muscle injury, regeneration, and repair. A re-view on the effectiveness of interventions to prevent ortreat muscle injury is outside the scope of this article,but some possible clinical implications of the informa-tion will be discussed.

Anatomy The myofiber or muscle fiber is the fundamental cellularunit of skeletal muscle: it is a multi-nucleated syncytiumformed by fusion of myoblastic cells10, 12. This myofibercontains the myofibrils, which are composed of sarcom-eres, arranged in series13-15. A sarcomere is the smallestcontractile unit of the muscle; it is composed of two types

Muscle Injury, Regeneration, and Repair / 9The Journal of Manual & Manipulative TherapyVol. 9 No. 1 (2001), 9 - 16

Fig. 1: Sarcomere. In: Huijbregts PA, Clarijs JP.Krachttraining in revalidatie en sport. Utrecht: De TijdstroomBV, 1995.

of proteins: contractile and structural proteins15 (Figure1). The contractile proteins make up the thick and thinfilaments. The thin filament consists of two intertwined

filamentary or F-actin chains. Tropomyosin and tropo-nin-C, -I, and -T are located in the groove between thesetwo chains. Myosin is the main protein of the thick fila-ment; it consists of two myosin heavy chains (MHC) andfour myosin light chains (MLC). The thick filament isformed by aggregation of these myosin molecules14, 15. Oneway to distinguish between the different fiber types is byidentifying the different isoforms of MHC and MLC thatthey contain15. Cross-striation of skeletal muscles is the result of anorderly arrangement within and between sarcomeres andmyofibrils. Maintaining this orderly arrangement is therole of the structural proteins. The myofibrils are alignedparallel to the long axis of the muscle fiber; the thin filamentsare anchored on either side of the sarcomere to a trans-verse structure called the Z-disc. In the Z-disc, each thinfilament is connected to four thin filaments from the adjacentsarcomere by a protein called alpha-actinin13-15. In turn,alpha-actinin is thought to be anchored in the Z-disc bythe proteins zeelin-1 and zeelin-21. The Z-disc containsthe proteins desmin, filamin, synemin, and zeugmatinat its circumference10. Desmin is called an intermediatefilament: its 8-12 nm diameter places it in size betweena smaller and a larger group of myofibrillar proteins3. Itmaintains inter-myofibrillar orientation by linking theZ-discs of adjacent myofibrils, and it stabilizes mitochondriaand nuclei to the sarcolemma3, 9. The Z-disc structure ofthe slow-twitch (ST) fibers is more complex and thickerthan that of the fast-twitch (FT) fibers10, 13-15; this struc-ture may play a role in the selective FT fiber injury ob-served after eccentric exercise3. The M-line is anothertransverse structure, located in the middle of the thickfilament. In this M-line, M-protein runs perpendicular

to the myosin molecules and maintains their spatialorientation. Myomesin anchors the protein titin to theM-line13-15. Intermediate filaments bridge the axis of themuscle fiber connecting adjacent M-lines3. C-lines aretransverse structures composed of C-protein: this pro-tein again stabilizes the thick filament arrangement13.Nebulin and titin are two proteins oriented parallel tothe thick and thin filaments. Titin runs from the Z-discto the M-line and is hypothesized to have a role in re-storing acto-myosin contact when no overlap exists af-ter excessive stretch; it also keeps the thick filament centeredin the sarcomere13. Sarcoplasmatic reticulum (SR) is an intracellullarstructure that sequesters and releases the Ca2+-ions neededfor acto-myosin interaction. In FT fibers, the SR embracesevery individual myofibril; in ST fibers, it may containmultiple myofibrils. The ATP-dependent enzyme calcium-ATP-ase plays an important role in pumping Ca2+-ionsback from the muscle fiber cytoplasm (or sarcoplasm)into the SR. ATP-depletion and subsequent decreasedcalcium-ATP-ase function may play a role in the Ca2+-induced autolysis discussed below. The transverse tubu-lar (TT) system consists of invaginations of the musclefiber membrane closely associated with the SR: actionpotentials are rapidly propagated throughout the musclefiber by way of this TT-system15. The muscle fiber membrane (or sarcolemma) is a lipidbilayer structure16. Small invaginations or caveolae al-low for stretch of the sarcolemma15. The protein dystrophin,lacking in patients with Duchenne muscular dystrophy,plays an essential role in mechanical strength and sta-bility of the sarcolemma1. Extensive membrane infold-ing and interdigitation with the extracellular CT increasethe surface area of the myotendinous junction (MTJ), onaverage, with a factor of 10 to 20. Infolding at the MTJof ST fibers is even more extensive, increasing surfacearea about 50-fold16. The MTJ has visco-elastic mechani-cal characteristics; the increased ST fiber infolding maybe in response to the creep experienced by the MTJ ofthese fibers during prolonged low-load contractions. Thedifference in MTJ surface area may be yet another expla-nation for selective FT fiber injury as a result of tensileforces16. The membrane infolding also places the mem-brane and the interface between membrane and extra-cellular CT at a very low angle (near zero) relative to thetensile force developed by the myofibrils (Figure 2). Thesarcolemma appears to be more resistant to the result-ant shear forces than to the tensile forces that would beproduced if the sarcolemma were oriented more perpen-dicular to the myofibril. The interface between sarcolemmaand extracellular CT also seems more resistant to shearforces. Muscle atrophy increases the angle of the mem-brane-terminal filament interface exposing it to more tensileforces; this may explain failure of the MTJ after immo-bilization-induced disuse atrophy16. Despite these adap-tations, the MTJ is very vulnerable to tensile failure4, 9,16,17:

10 / The Journal of Manual & Manipulative Therapy, 2001

Fig. 2: Schema of the structures involved in force trans-mission between tendon and contractile proteins of musclecell. Extracelluar components (EX) include tendon col-lagen fibers (1) and basement membrane (2). The junc-tional plasma membrane (3) separates extracelluar (EX)and intracelluar (IN) force-transmitting structures. Withinthe cell, thin actin filaments (5) are attached to the cellmembrane by dense, subsarcolemmal material (4). Copy-right 1987 American Academy of Orthopaedic Surgeons.Reprinted from Garrett W, Tidball J. Myotendinous Junc-tion: Structure, Function, and Failure. In: Woo SLY, BuckwalterJA. Injury and repair of the musculoskeletal soft tissues.AAOS, Rosemont, IL, 1987.

a predilection for a tear near the MTJ has been reportedin the biceps and triceps brachii, the rotator cuff muscles,the flexor pollicis longus, the peroneus longus, the medialhead of the gastrocnemius, the rectus femoris, the ad-ductor longus, the iliopsoas, the pectoralis major, thesemimembranosus, and the whole hamstrings group2, 18.

The terminal sarcomeres near the MTJs are shorterthan the more centrally located sarcomeres: this allowsfor increased speed of contraction but decreased force16.It has been hypothesized that the increased contractionspeed allows for pre-loading of the MTJ before the othersarcomeres reach peak tension16. Muscle contraction (andthus possibly MTJ pre-loading) results in greater forceand energy absorbed prior to failure18. The distance ofthese terminal sarcomeres to the neuromuscular junc-tion is greater, meaning that an action potential has totravel further along the sarcolemma and the TT-system.Shorter terminal sarcomere length may allow for a moreuniform tension development along the whole length ofthe muscle fiber rather than earlier tension development15.Hypotheses with regards to the role of the shorter ter-minal sarcomeres in causing muscle fiber injury arediscussed below. Filamentous actin connects the Z-discsof the terminal sarcomeres to sub-sarcolemmal densi-ties at the MTJ. The proteins vinculin and talin connectthese actin filaments to the sarcolemma of the MTJ. Theterminal Z-discs are also directly structurally continu-ous with these densities16. The basement membrane is a loose matrix of glyco-proteins and collagen fibers located outside the sarco-lemma14. At the MTJ, the large transmembrane glyco-

protein integrin may link the proteins vinculin and talin(discussed above) to the basement membrane16. Thebasement membrane consists of a basal and a reticularlamina. The basal lamina is located closest to the celland consists of an inner lamina rara and an outer laminadensa10; the reticular lamina merges with the endomy-sium along the length of the fiber; at the MTJ, it mergeswith the collagen fibers of the tendon16. The protein laminin,the glycoprotein fibronectin, and type IV collagen havebeen hypothesized to play a role in this basement mem-brane-tendon interaction16. The basement membrane hasan important mechanical function: it dissipates up to 77%of the energy lost during a stretch-shortening cycle ofthe muscle1. It may seem illogical to introduce an en-ergy-dissipating structure such as the basement mem-brane in a chain of structures whose function is to transmitforce. However, a perfectly elastic system may sufferoscillation-induced tensile strain; the basement membraneprovides for functionally necessary viscous energy lossand thus protects the muscle fiber and tendon16. A CT sheath called the endomysium encloses indi-vidual muscle fibers; groups of fibers are enclosed by theepimysium. The perimysium encloses the whole muscleand is directly continuous with the deep fascia10, 15. Be-yond the physiological length of the muscle, actin-myo-sin overlap is lost: here these CT sheaths (together withsome of the structural proteins and the basement mem-brane) are responsible for resisting tensile force18. Dam-age to these sheaths is more likely in multi-joint muscles,which may be exposed to greater stretch. Intact CT sheathsmay also act to bypass tension past dysfunctional or scarredzones of the muscle fiber9. The endomysium is less well-developed in FT fibers, possibly again making them moresusceptible to stretch-induced injury17. Blood vessels supplying the muscle run in these CTsheaths between muscle fiber bundles. They course atright or oblique angles through the perimysium. In theepimysium, they form a capillary network around theindividual fibers. The capillaries have a tortuous coursewhen the muscle is contracted, and they straighten outwhen the muscle is extended10

As noted earlier, muscle fibers are a multi-nucleatedsyncytium formed by the fusion of mono-nucleatedmyoblasts: the muscle fiber nuclei are located peripher-ally in the muscle fiber just below the sarcolemma15. Duringmyogenesis, a subpopulation of myoblasts is not incor-porated into this syncytial structure. These myoblastsbecome the satellite cells and are located outside of thesarcolemma, but inside the basement membrane. Throughmitosis, these cells provide the myonuclei needed forpostnatal growth. At maturation, the satellite cells be-come mitotically quiescent. The important role satellitecells have as stem cells providing the myoblasts neededfor muscle regeneration is discussed below. FT fibers havefewer satellite cells than do ST fibers12. With age, the numberof satellite cells decreases; the effect of these factors on

Muscle Injury, Regeneration, and Repair / 11

the ability for full regeneration after muscle injury isunknown15.

Muscle injury mechanisms The introduction notes that muscle injury may re-sult from mechanical stress, thermal stress, myotoxic agents,and ischaemia. The next section discusses the probablecentral event in muscle injury, the loss of calcium ho-meostasis1. There are three groups of hypotheses regardingthe initial event causing this loss of calcium homeosta-sis: metabolically induced, temperature induced, ormechanically induced1,19.

Metabolic hypotheses Metabolic hypotheses concentrate on two mechanisms:ATP-depletion and the production of oxygen free radi-cals. Depletion of ATP might result from increased metabolicdemands due to exhaustive exercise or ischaemia11. Low-ered intracellular ATP-concentrations will decrease cal-cium-ATP-ase activity and cause SR dysfunction1. A pro-gressive depression of Ca2+-uptake has been shown withincreased duration of low-intensity exercise, but also withshort duration high-intensity exercise20. The inability tosequester calcium at an appropriate speed will slow musclefiber relaxation, and slowed relaxation will affect mechanicalproperties of the myofibrils resulting in a functional rigor.If this rigor is confined to only some myofibers or myo-fibrils, the resultant shear forces between adjacent musclefibers and myofibrils during continued muscle contrac-tion and relaxation may cause mechanical interfiber andinterfibrillar disruption, respectively9,17. SR dysfunction will increase cytosolic Ca2+-concen-trations. Even physiological concentrations have been shownto result in autolytic degradation. Duration of increasedCa2+-concentrations and biological availability to activateautolytic enzymes may explain why these physiologicalconcentrations do not initiate autolysis in vivo: Ca2+-concentrations are transient due to continuous bindingto troponin-C and subsequent re-uptake in the SR1. However,the Ca2+-pool in the SR has been shown to be sufficientfor initiating autolytic breakdown, even in the absenceof sarcolemmal disruption20. SR dysfunction initiatingautolysis resulting in delayed loss of sarcolemmal integ-rity may explain why blood levels of muscle enzymesindicating sarcolemmal damage usually only increase aminimum of 24 hours after a muscle injury inducing exercisebout21. Arguing against this ATP-depletion hypothesis is thefact that global cytosolic ATP-concentrations remain nearresting level despite exhaustive exercise. However, thisdoes not exclude compartmental decreases in ATP-con-centration. The lower metabolic cost of eccentric exer-cise when compared to concentric exercise makes the ATP-depletion hypothesis an unlikely explanation for muscle

injury induced by eccentric strength training regimens1.Teague and Schwane22 argued against a metabolic expla-nation for this type of injury: they had subjects performten repetitions of eccentric elbow flexion at 60% of theirmaximal isometric force. Subjects were given no rest, 15seconds, 5 minutes, or 10 minutes between repetitions,but all had symptoms of post-exercise muscle-injury favoringan explanation of cumulative mechanical strain ratherthan a metabolic cause. The ATP-depletion hypothesismay, however, play a role in explaining the predilectionfor FT fiber damage with high-repetition, low load ec-centric endurance-type regimens as observed by Fridenet al23. It may also explain the selective FT fiber damageresulting from ischaemia seen by Lieber et al11. With thistype of eccentric exercise (or tourniquet-induced ischaemia),the lower oxidative capacity of FT fibers may result inmore rapid substrate depletion, subsequent functionalrigor, and mechanical damage3, or alternatively loss ofcalcium homeostasis and autolysis without mechanicaldamage. A second metabolic hypothesis concentrates on anincreased production of free oxygen radicals. Superox-ide anion and hydrogen peroxide are free oxygen radi-cals produced as a common metabolic intermediate inhighly active tissues20. Free radicals can oxidize phos-pholipids, carbohydrates, and proteins1,20. Membrane lipidperoxidation may result in sarcolemmal disruption andsubsequent rapid calcium influx disturbing the calciumhomeostasis1. The pump function of calcium-ATP-ase isimpaired by free radical oxidation of its sulfhydryl groups20.Free oxygen radical production increases during exer-cise19. The lower metabolic cost of eccentric exercise hasbeen hypothesized to cause a relative overperfusion withincreased sarcoplasmic pO2 favoring the production offree oxygen radicals1. Mechanical disruption of the cy-toskeleton stabilizing the organelles in the muscle fiberhas also been hypothesized to alter the position of themitochondria and physically disrupt the association ofthe components of mitochondrial respiration resultingin increased free radical production in the mitochondrialelectron transport system1. Arguing against this free radicalhypothesis as an explanation for eccentrically inducedinjury is the fact that the metabolic cost for eccentricexercise is lower than that of concentric exercise. Also,overperfusion during eccentric exercise has not beendemonstrated. Free radicals may, however, play a role inother types of muscle injury.

Temperature hypotheses Increased muscle temperature is usually associatedwith decreased injury potential: it reduces gamma fiberactivity and sensitivity of muscle spindles to stretch,enhances enzymatic function, decreases viscosity, andincreases CT extensibility18. However, increased temperaturemay also increase the rate of unwanted structural lipid


and protein degradation. Increased sarcolemmal viscos-ity may bring the enzyme phospholipase A2 into contactwith its phospholipid substrate in the plasma membraneresulting in sarcolemmal degradation1. Increased tem-perature may negatively affect calcium-ATP-ase function20.Eccentric muscle activity may result in increased tem-perature, as some of the energy absorbed by the muscleis dissipated by heat. Relatively lower heat removal (dueto a lack of vasodilation) may raise intramuscular tem-perature1. The temperature hypothesis may also play arole in thermal muscle damage and muscle injury due toconcentric exercise.

Mechanical hypotheses A muscle fiber will fail if the tensile or shear stressinduced in a structural component exceeds its yield strength.This stress may be the result of a single mechanical eventor of cumulative, repetitive, mechanical events1. Multi-joint muscles can be subject to greater stress and are,therefore, at greater risk for mechanically induced fail-ure1. In addition, the thinner and less complex Z-disc,the smaller MTJ surface area, and the less well-developedendomysium put FT fibers at greater risk for tensile failure.FT fibers are capable of higher speed and force produc-tion than ST fibers resulting in higher tensile stress1,15.Forces produced during eccentric muscle action may exceedmaximal isometric force by 50-100%1. There is someevidence that FT fibers are preferentially recruited foreccentric muscle action24. As discussed earlier, their loweroxidative capacity may put the FT fibers at further riskfor damage. Eccentric exercise has been reported to se-lectively damage FT fibers3,23. However, eccentric exer-cise may also cause ST fiber injury: Mair et al25 foundincreased blood levels of a MHC isoform found in ST fibersafter 7 sets of 10 repetitions of eccentric quadriceps activityat 150% of maximal voluntary strength. Increased velocity of eccentric lengthening may de-crease the number of acto-myosin cross-bridges avail-able to resist the external force. This increases the forceper active cross-bridge predisposing the contractile pro-teins to mechanical disruption1. Metabolically inducedfunctional rigor may cause non-homogenous lengthen-ing of adjacent sarcomeres or myofibrils: shear stressescan damage desmin and other intermediate interfibril-lar proteins causing a disruption of myofibrillar arrange-ment1,3. Improper interdigitation of thick and thin fila-ments after overstretching a sarcomere weakens thesarcomere and may add stress on other sarcomeres orthe adjacent SR and sarcolemma; the sarcolemma hasbeen shown to bear most of the passive tension at sar-comere lengths over 140-150% of resting length1. Ex-cessive sarcomere lengthening may also damage thelongitudinally oriented proteins titin and nebulin, explainingthe observed axial misalignment of thick filaments aftereccentric exercise3.

The apparent predilection of muscle injury for theMTJ occurs regardless of muscle architecture, directionof force, and whether the muscle was contracted or pas-sively stretched4,9. Differences in sarcomere length affectcontractile speed and force3,15; this results in differentforces being transmitted to the Z-disc where shorter andlonger sarcomeres are located next to each other in se-ries. The undue directional stress may mechanically dis-rupt the Z-disc. MTJ injury may be a result of the shorterterminal sarcomeres joining up with the longer morecentrally located sarcomeres3.

Stages of muscle injury, regeneration, and repair Independent of injury mechanism, the pathophysiologi-cal events in muscle tissue regeneration and repair arevery similar8. The four stages of this process are Ca2+-overload, autolysis, phagocytosis, and regeneration/re-pair1.

Ca2+-overload As discussed earlier, loss of intracellular calciumhomeostasis is central to muscle injury, regardless of whetherthe injury mechanism is metabolic, thermal, mechani-cal, or, as is most likely, a combination of these injurymechanisms. The importance of maintaining cytosolicfree Ca2+-concentrations within narrow margins is indi-cated by the large number of mechanisms the musclefiber has for transporting Ca2+ out of the sarcoplasmiccompartment1. There are multiple ways in which cyto-solic calcium concentration can increase. The extracel-lular free Ca2+-concentration is 2-3 millimol/l versus theintracellular concentration of 0.1 micromol/l of a rest-ing muscle fiber. Disruption of the sarcolemma wouldallow Ca2+ entry into the muscle fiber down its electro-chemical gradient1. Dysfunction of the SR, whethermetabolically, mechanically, or thermally induced, mayalso increase cytosolic calcium concentrations, sufficientfor initiating autolysis1,20. Voltage-dependent Ca2+-chan-nels are located in the TT-membranes; stretch-sensitiveCa2+-channels may be mechanically opened during length-ening contractions. Calcium channel blockers have beenshown to attenuate exercise-induced muscle injury, butthe importance of these channels is considered minimal1,19.Several muscle fiber receptors act to release Ca2+ frominternal stores or allow for interstitial influx; some ofthese receptors respond to histamine and bradykinin1.In the Ca2+-overload phase, the calcium-transport andbuffering mechanisms are overwhelmed, resulting in in-creased intracellular Ca2+-concentrations and subsequentautolysis1.

Autolysis Autolytic mechanisms are initiated if the sarcoplas-


mic Ca2+-concentration is sufficiently elevated for a suf-ficient period of time. Armstrong et al1 call this stage theautogenetic stage. Though not a proteolytic event, in-creased Ca2+-concentrations may cause uncontrolledcontraction of sarcomeres in the affected region; this servesto wall off the injured area during subsequent stages, butit also increases tensile forces and may increase damage.Mitochondria can buffer elevations in cytosolic calciumlevels. The mitochondria of ST fibers buffer Ca2+ at a ratetwo to three times that of FT fiber mitochondria. Intra-mitochondrial accumulation in the nanomolar range actuallyincreases function, but accumulation in the micromolarrange depresses mitochondrial function. MitochondrialCa2+-overload may result in ATP-depletion1. Calpains areCa2+-dependent intramuscular proteases, which, whenactivated by elevated calcium concentrations, can cleavemyosin, alpha-actinin, talin, and vinculin1,7,20. The pri-mary form of the enzyme phospholipase A2 (PLA2) is Ca2+-dependent1,19. PLA2 is located in the sarcolemma, organellemembranes, sarcoplasm, and intracellular lysosomes. Itdamages the cell membrane by using membrane phos-pholipids as a substrate for the production of arachidonicacid and subsequently prostaglandins, leukotrienes, andthromboxanes1. PLA2 is the main active ingredient in snakeand bee venom1. Prostaglandin E2 (PGE2) is one of theproducts of PLA2. PGE2 stimulates the activity of proteolyticenzymes contained in intacellular lysosomes; these ly-sosomal proteases may play a role in degrading myofibrillarproteins1. The amount of intramuscular lipofuscin gran-ules increases after eccentric exercise. Lipofuscin is anindigestible residue of lysosomal degradation, support-ing a role for these proteolytic enzymes in exercise-in-duced muscle damage3.

Phagocytosis The autolytic activity continues into the phagocyticphase1. Ca2+-induced hypercontraction of disrupted andretracted myofibrils isolates the injured area27. By thesecond day after the injury, this hypercontracted bandmakes place for a membranous structure, the demarca-tion membrane, which then walls off the necrotic area5.Neutrophil leucocytes, macrophages, and T-lymphocytesinvade the injured area. However, phagocytic cells arethe prominent feature of this stage: leucocyte concen-trations decline within 24 hours and only 30% of the T-lymphocytes present in the wound area are actually ac-tivated7. Armstrong et al1 reported that the phagocyticphase starts within two to six hours. Garrett2 reportedan invasion of inflammatory cells by one to two days. Russellet al6 noted macrophage infiltration within 24 hours, andReid4 stated that phagocytosis is ongoing by 48 hoursafter injury. Macrophage infiltration only occurs whenthe necrotic area is (re) vascularized10; this may explainthe discrepancies noted with regards to the start of thephagoytic stage. We can expect that injuries compromising

circulation, such as ischaemic injuries, take longer toenter this phagocytic phase. The inflammatory and phagocytic cells may be theresult of activation and mitosis of quiescent cells alreadypresent in the muscles7. St. Pierre and Tidball26 describedthe presence of macrophages in the tendon of rats nearthe MTJ, which increased their synthetic activity in re-sponse to altered mechanical demands on the MTJ. However,the majority of cells probably result from chemotaxis outof the circulatory system7. The substance providing thestimulus for this chemotaxis is unknown. Injured musclereleases a number of substances into the extracellularspace; basic fibroblast growth factor (bFGF), transferrin,and an uncharacterized mitogen all have mitogenic functionson myogenic cells. Platelet-derived growth factor (PDGF)is also released by injured muscle. PDGF is a mitogenand chemo-attractant for both inflammatory cells andfibroblasts. However, PDGF has a very short half-life. Thedelay between injury and arrival of inflammatory cellsmakes it likely that the substances released serve to activatethe resident macrophages and fibroblasts. These macroph-ages, already present in the muscle, then secrete trans-forming growth factor beta (TGF-beta), interleukin-1 (IL-1), and PDGF, all chemo-attractants to inflammatory cells7. In addition to removal of cellular debris and phago-cytosis, the inflammatory cells regulate the start of muscleregeneration7. Macrophages secrete insulin-like growthfactor (IGF), bFGF, and TGF-beta6,27. These substancesactivate both satellite cell proliferation and myoblastdifferentiation, and cause the expression of embryonicMHC7,27, the MHC-isoform seen initially in regeneratingmuscle26,28. NSAIDs are commonly used after injury, in-cluding muscle injury; however, these medications maybe contra-indicated as a result of their inhibition ofmacrophage and neutrophil function. Tidball7 reportedthat using NSAIDs resulted in slower regeneration andweaker MTJs. Damage to the basement membrane mayalso play a role in the activation of satellite cells by ex-posing these cells to the mitogen-rich extracellular matrix28.

Regeneration and repair There are two distinct ways in which a muscle canrespond to injury: regeneration and repair. Regenerationinvolves complete restoration of the pre-injury structure.With repair, the production of a CT scar may restore musclecontinuity but at the same time inhibit complete regenera-tion. There is the mistaken notion, even in some recentmedical literature, that skeletal muscle does not have thepotential to regenerate8,10. After activation by factors excreted by the macroph-ages and/or exposure to the mitogen-rich extracellularenvironment as a result of a lesion to the basementmembrane, satellite cells start to proliferate and differ-entiate into myoblasts5. There is no satellite cell recruit-ment from adjacent muscles12. Schultz12 noted that sat-


ellite cells migrate to the injury site from all along themuscle fiber. In contrast, Hurme and Kalimo27 stated thatmigration of satellite cells plays a minor role; most myoblastsmay be produced near or at the injury site. By three dayspost-injury, these myoblasts fuse and form a syncytialmyotube5. Myotubes are characterized by a centrally locatedchain of enlarged nuclei, prominent nucleoli, and abun-dant polyribosomes upon which muscle proteins aresynthesized10. The myosin produced initially contains anembryonic MHC-isoform, which is replaced later by adultisoforms26,28. The contractile proteins are first assembledinto well-organized bundles of thick and thin filamentsat the periphery of the myotube. Later myofilaments areadded successively towards the center of the myotube.Construction of the TT-system and SR occur simultaneouslywith the assembly of the myofilaments. Breaking up thecentral chain of nuclei and subsequent migration of thesenuclei to a position near the sarcolemma marks the tran-sition from myotube to muscle fiber10. There are two theorieswith regards to the formation of growth extensions atthe end of the damaged muscle fibers. The continuoustheory holds that muscle is repaired by outgrowth of thedamaged myoplasm with migration of myonuclei towardsthe damaged muscle fiber stumps. The discontinuous theorystates that myoblasts fuse, form myotubes, and then fusewith the muscle fiber stumps. Robertson et al29 foundevidence supporting the latter theory in their experimentsin mice. The myoblasts also produce a highly hydrated extracel-lular matrix. Initially, hyaluronic acid is the main con-stituent of this matrix, but this is later replaced by a muscle-specific chondroitin-sulphate proteoglycan (M-CSPG).Anchored at the cell surface, M-CSPG ensures sufficientspace for subsequent increases in muscle fiber girth byincreasing volume through binding water electrostati-cally. M-CSPG is finally replaced by heparan-sulphateproteoglycans; these insert into the cell membrane andform part of the new basement membrane10. By day 7, anew basement membrane forms within the old one5. Myotubes may release chemo-attractants for vascu-lar endothelial cells, thus promoting adequaterevascularization10. Complete rupture of a myofiber dis-connects part of the fiber from the neuromuscular junc-tion. Regeneration occurs independently of innervation.Final differentiation into the different muscle fiber typesdoes, however, depend on reinnervation of the regener-ating denervated portion of the muscle fiber9. Optimal regeneration is facilitated by an intact base-ment membrane, an uninterrupted vascular supply, anda functional nerve17. An intact basement membrane keepsout fibroblasts and a majority of newly forming collagenfibers5. Re-growth of damaged muscle fibers is obstructedby excessive, interposed CT5. Collagen deposition in theinjury zone may prevent longitudinal fusion and resultin split muscle fibers sometimes found after injury29. Withcontusion, laceration and strain CT sheaths may be dis-

rupted along with muscle fibers5. Bleeding occurs afterthis type of injury; this bleeding may escape through theperimysium and fascia and dissipate into the subcutane-ous space, but it may also remain contained within themuscle substance2. This intramuscular haematoma isreplaced by proliferating granulation tissue, which maymature into a CT scar5. Extensive CT proliferation is foundespecially in crush injuries27. Tensile forces introducedin the injured fiber prematurely may re-injure the fiber,cause renewed bleeding, and result in excessive CT depositionand repair rather than regeneration. Jaervinen and Lehto8

caused a partial rupture in rat gastrocnemius muscle.Immediate mobilization resulted in increased bleeding,more abundant inflammatory cell infiltration, a more parallelorientation of the regenerating myofibers, increasedcapillarization, but also more extensive proliferation ofCT in the injured area. Immobilization led to a more irregularorientation of muscle fibers, but less CT proliferation.Immobilization also greatly decreased force to failure,elongation at failure, elastic stiffness, and energy absorptioncapacity. Mobilization, on the other hand, quickly restoredthese parameters to control values. Short immobiliza-tion times decreased CT proliferation as compared toimmediate mobilization. After two days of immobiliza-tion, remobilization resulted in re-rupture; no rupturewas noted after five days of immobilization. Two days ofimmobilization resulted in similar elastic stiffness val-ues as in muscles immediately mobilized.

Implications An in-depth review of the effectiveness of the inter-ventions used for prevention and treatment of muscleinjury is outside the scope of this article. However, it ispossible to derive some theoretical implications from thematerial presented. Atrophy predisposes the MTJ to fail-ure: careful progressive resistance exercise after immo-bilization resulting in disuse atrophy may restore thesarcolemma-tendon angle and prevent injury associatedwith more aggressive remobilization. Excessive stretchas may occur especially in multi-joint muscles has beenassociated with tensile muscle failure. A stretching regi-men may adapt the length of these multi-joint musclesto the excursion required during sport and ADL tasks. Awarm-up may also prevent tensile injury by increasingCT and muscle extensibility, by decreasing unwanted tensionincreases as a result of overactive stretch reflexes, andby increasing enzymatic and thus muscle function. Highmuscle temperatures may initiate muscle injury. Athletesshould probably not work out in extremely high tempera-tures. Cardiovascular exercise increases capillarizationand may allow for increased dissipation of excess heatproduction. Endurance-type exercise may also play a rolein prevention of muscle injury as a result of high-repeti-tion low load eccentric exercise: fiber type conversioncan increase muscle fiber oxidative capacity and allows


the muscle fibers to better deal with high metabolic de-mands. Of course, technique training and the resultantimproved neuromuscular coordination may prevent musclesand CT from ever being stressed to the point of failure. Theoretically, muscle regeneration restores contrac-tile characteristics to a greater extent than does musclerepair. Immediate mobilization rapidly restores mechani-cal characteristics but also results in greater CT prolif-eration. A short period of post-injury mobilization tem-porarily depresses tensile characteristics but results in lessCT deposition and more complete regeneration. However,short-term immobilization may predispose the muscle to

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