**22. The role of lactate in the process of muscular healing**

by the fact that the satellite cells and the necrotic parts of the injured muscular fibers, release several substances defined "wound hormones", which behave as chemo-attractant increasing in such a way the overflowing of the inflammatory cells (Tidball, 1995; Chazaud et al., 2003; Hirata et al., 2003;). Inside the injured muscle, we may observe macrophages and fibro-blasts whose activation gives origin to the additional chemo-tactic signals (as growth factor, chem‐ iochins and citochines) directed at the circulating inflammatory cells. In addition to this quota of growth factors, produced *ex-novo*, the main part of the muscle tissue contains growth factors stocked in active form inside its ECM, ready to be used in cases of urgent necessity; like for example in the repair of a lesion. (Ragk and Kerbel, 1997). In the case of tissue injury the capacity of biological tissue repair depends on the release of the activation of the growth factors ECM-dependent (or to the growth factors tied to the ECM) and of their capacity to start repair processes (Ragk and Kerbel, 1997). In particular, direct evidence exists that the Tumor Necrosis factor-α (TNF-α) covers and important physiological role in the repair process of the injured skeletal muscle, which is shown by the fact that, if its activity is inhibited during the healing process, there is a slight deficit of the repair capacity of the skeletal muscle itself (Warren et al., 2002). In addition, a large number of growth factors and citochine, as member of the family of Fibroblastic Growth Factors (FGF) of Insulin-Like Growth Factors (IGF), and of the family of Transforming Growth Factors-β (TGF-β), the Hepatocyte Growth factors (HGF), the Interleukin 1β (IL-1β) and the Interleukin-6 (IL-6), are amply known for their expression during muscular injury. After all it is also certain that many other factors, like the Plateled –Derived Growth-Factors are present in the course of various stages which are registered in a muscular injury (Mishra et al., 1995; Burkin and Kaufman, 1999). We should also note the fact that their expression may be induced, in the field of the skeletal muscle by physiological stimuli similar to those which cause micro-traumatic lesions, such as the phenomena of overstretching, or those relative to non-appropriate external mechanical loads (Burkin and Kaufman, 1999; Perrone et al., 1995). Considering the fact that these growth factors make up powerful mytogenic activators for numerous types of cells, it is now an acquired fact that the latter may also be involved in the activation of regenerative processes of the injured skeletal muscle (Burkin and Kaufman, 1999; Best et al., 2001; Chargè and Rudnicki, 2004). A certain number of these growth factors, like FGFs, IGF1, IGF2, TGF-β, HGF. TNF-α and the IL-6,are potential activators of the proliferation of the myogenic precursor cell (MPC,Myogenic Precursor Cells or satellite cells) (Chargè and Rudnicki, 2004). Some of these are also powerful stimulators for the differentiation of the MPC and after, in the course of regenerative processes, regarding the fusion of myotubes in multi-nuclear mature myo-fibers (Burkin and Kaufman, 1999; Best et al., 2001; Chargé and Rudnicki, 2004). In the acute phase, following a harmful muscular event, the polymorphonuclear leukocytes are the most abundant cells present on the injured area (Hurme et al., 1991; Thorsson et al., 1998; Brickson et al., 2001; Schneider et al., 2002; Bricksona et al., 2003) but, before the first day, the latter are substituted by the monocytes. In relation to the basic principles of an inflammatory process, these monocytes are eventually transformed into macrophages, which are employed in proteolysis and phagocytosis of the necrotic material, thanks to the release of lysosomal enzymes (Hurme et al., 1991; Best and Hunter, 2001; Farges et al., 2002 ; Timballi, 1995) The phagocytosis on behalf of the macrophages depending on the necrotic material, makes up a highly specific process. In this phase the intact

32 Muscle Injuries in Sport Medicine

A few hours from the injury, the consumption of oxygen at rest, inside the injured muscular area, rises drastically, generating as a consequence an imbalance between the storing and the request of O2, which in its turn determines a rapid descent in the tension of O2 inside the injured area. Contextually to this, we assist in an increase in the concentration of lactate inside the lesion. All this series of events is well shown in the process of repair tissue in the ear of the rabbit observed at 15 days from the traumatic event(Hunt and Hussain, 1993) At the moment in which the tension of the O2 falls, the process of accumulation of lactate starts (Wasserman et al., 1990); to this end it is important to remember that the muscle produces a superior quantity of lactate than that which it consumes, in all conditions, including at rest (Graham et al., 1986). In this physiological context, the lactate assumes a sort of "guide role", inducing the fibroblasts to produce collagen and influencing the macrophages, and eventually also the lymphocytes, to excrete angiogenic substances. The repair components which we could describe as "lactate-guided" would seem to assume an even further importance, above all at the moment in which the inflammatory component diminishes notably, or starting, approxi‐ mately, from the seventh post-lesion day (Hunt and Hussain, 1993). The accumulation of lactate in the injured area is substantially ascribable to three factors. The first of these is made up of the fact that the vascular damage, following the tissue damage, inhibits the diffusion of O2 inside the injured tissue, from this follows a quota of lactate which is produced by anaerobic glycolysis (Im and Hoopes, 1970a: 1970b). The second of these, the vascular damage limits the external diffusion of lactate (Hunt et al., 1967) and the last reason, fact which makes up the most important aspect, is the activation of the leucytes which causes the release of a large quantity of lactate, both of hypoxic nature and not (Calwell et al., 1984). The macrophages which appear on the site of lesion a few hours before the harmful event- playing the role of "guided cells" in the field of the first repair processes they are not only able to supply the injured area with lactate, but are also influenced by the quota of lactate present. In fact, confirming this hypothesis, it is possible to note how the concentration of lactate inside the injured area, diminishes only slightly at the moment in which the concentration of O2 rises (Hunt et al., 1978). To this end, it is worth it to mention how some authors report values of lactate concentration, inside the injured muscle, between 8 and 18 mmol.1-1 (Hunt and Hussain, 1993). On the other hand, the hypothesis, that lactate was implied in the synthesis of collagen, it had already been put forward by some authors more than forty years ago (Green and Goldberg, 1963; Levine and Bates, 1976). In these experiments, it was described how lactate was implied in the synthesis of collagen, the authors noted how in their experiments the fibroblats put into culture, produced a higher quantity of collagen, in comparison with the control group when the concentration of lactate surpassed 20mmol.1-1. In these, just as in other successive experiments of such kind, it was observed in a hypoxic regime, the production of collagen is delayed up until the moment in which the hypoxic cells are not supplied with oxygen. In other words, the production of collagen only starts when there is the contextual presence of oxygen and lactate. This data suggests how the effect of lactate is independent in comparison to that of oxygen (Comstock and Udenfriend, 1970). However, in spite of the first stimulating results, this line of research has been practically abandoned since 1976. After nearly 20 years, other authors speculated that the lactate could work as a regulator in the process of collagen synthesis inside the injured tissue (Hunt and Hussain, 1993). According to these authors, the maximum ratio of collagen production, would occur in the presence of a high concentration of lactate, included between 8 and 18 mmol.1-1, concurrent with an high value of PO2, equal to about 100 mm Hg. This data, at a first look, would seem paradoxical, since we can usually consider logical a strong presence of lactate where there are scarce conditions of O2. However, we need to remember that the leucocytes are responsible for the production, in aerobic conditions, for an important quantity of lactate inside the injured tissue area, and that the production of lactate on behalf of the leucocytes inside the injury remains high also in the presence of a high value of PO2 (Levine and Bates, 1976). This type of biological model, characterized by a high concentration of contextual lactate to a high value of PO2, would establish a favorable condition, not only to the collagen synthesis but also to the angiogenesis (Hunt and Hussain, 1993) and in addition it is also probable that the lactate serves as stimulus for the secretion of TGF-β in the injured area (Falanga et al., 1991).

ly, a reserve pool of undifferentiated cells, defined satellite cells are, during the fetal development, dislocated under the basal lamina of each singular muscular fibre (Hurme and Kalimo, 1992; Rantenen et al., 1995; Kalimo et al., 1997;). In answer to the harmful event, these particular cells, initially proliferate, then differentiate into myoblasts and at the end of the process, they connect to the remaining fibers forming multinuclear myotubes. The multinu‐ clear myotubes of recent formation fuse, in a second moment, with the part of the injured fiber which survived the initial trauma (Hurme and Kalimo, 1992). In the end, the part of regener‐ ated myofiber acquires its mature form, with normal streaking and with the myonuclei peripherally dislocated (Hurme and Kalimo, 1992). Curiously, in answer to t very balad traumas, like for example in a singular eccentric elongation which provokes trauma of light entity, the satellite cells immediately respond starting to proliferate, but due to the limitation of trauma and of the rapid "innate" answer of repair on behalf of the fibres of the injured muscle, they auto-block their activation before myoblasts are formed (Aarimaa et al., 2004). In the mature skeletal muscle there exists at least two principle populations of satellite cells (Rantenen et al., 1995; Jancowski et al., 2002; Kalimo et al., 1997; Qu-Peterson et al., 2002;; Rouger et al., 2004; Zammit et al., 2004). The "classic" satellite cells which reside under the basal lamina of the muscular fiber and which can be divided into"committed satellite cells" which are ready to differentiate themselves into myoblasts immediately after the harmful event and the "stem sateliite cells" which have to first undergo cellular division to be able to differentiate (Kalimo et al., 1997; Rantenen et al., 1995; Zammit et al.,2004). Through this cellular division (which may be seen form a biological point of view as a true and proper proliferation process), the population of stem satellite cells, again builds up the reserve of satellite cells for a future possible regenerative request (Rantenen et al., 1995; Zammit et al., 2004). In this population of satellite cells, we may note the existence of and under-population of cells capable of differentiation, beyond the myogenic lines, not only in mesenchymal lines but also in neural or endothelial ones (Jankowski et al., 2002; Qu-Peterson et al., 2002). Up until to today the satellite cells were retained the only resource of the myonucleus in the course of muscular repair (Chargé and Rudnicki, 2004), recent discovery has shown the presence of a different population of multi-powerful stem cells, which can contribute to the reparation of the injured skeletal muscle; the "non-muscle-resident stem cells" (Chargé and Rudnicki, 2004). In fact, even some isolated progenitor cells of the bone marrow (BM), the neuronal compart‐ ment, and different mesenchymal tissue, are able to differentiate in myogenic lines. The cells derived from BM, not only contribute to the regeneration of the muscles fibers in the injured skeletal muscle, but they are also able to re-integrate the pool of the satellite cells in the injured skeletal muscle (Labarge and Blau, 2002). In each case, it is important to note that the frequency at which these events happen seems to be very low, also in the case of serious lesion, if compared to the number of regenerated myoblasts derived from the "muscle-resident" satellite cells (Grounds et al., 2002; Labarge and Blau, 2002). So, it is quite disputable the fact that the "non muscle-resident" stem cells may give a significant contribution to the repair of injured skeletal muscle (Ground et al., 2002) In addition to the classic satellite cells, resident in the lower part of the basal lamina, there also exists a distinct population of extra-lamina collocated stem cells, inside the connective tissue of the skeletal muscle (Dreyfus et al., 2004). In answer to a harmful event on the skeletal muscle, these cells take part in the formation of

Etiology, Biology and Treatment of Muscular Lesions

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35

#### **23. The repair and re-modeling phase of the muscular fibers**

Once the phase of destruction has dropped in intensity, the real repair process of the injured muscle begins, which shows itself through two concurrent processes, which show between themselves, at the same time, complementary and antagonistic: the repair of the destroyed myofibers and of their respective innervations and the formation of healing tissue. A balanced progression of these two processes, makes up an essential pre-requisite for an optimal reactivation of the contractile functions of the skeletal muscle (Kalimo et al., 1997; Hurme and Kalimo, 1991;). In spite of the fact that the muscular fibers are, in general, considered as fibers of irreversibly post-mitotic type, the notable repair potential of the skeletal muscle is guaran‐ teed by an innate mechanism able to reactivate the injured contractile apparatus. Consequent‐ ly, a reserve pool of undifferentiated cells, defined satellite cells are, during the fetal development, dislocated under the basal lamina of each singular muscular fibre (Hurme and Kalimo, 1992; Rantenen et al., 1995; Kalimo et al., 1997;). In answer to the harmful event, these particular cells, initially proliferate, then differentiate into myoblasts and at the end of the process, they connect to the remaining fibers forming multinuclear myotubes. The multinu‐ clear myotubes of recent formation fuse, in a second moment, with the part of the injured fiber which survived the initial trauma (Hurme and Kalimo, 1992). In the end, the part of regener‐ ated myofiber acquires its mature form, with normal streaking and with the myonuclei peripherally dislocated (Hurme and Kalimo, 1992). Curiously, in answer to t very balad traumas, like for example in a singular eccentric elongation which provokes trauma of light entity, the satellite cells immediately respond starting to proliferate, but due to the limitation of trauma and of the rapid "innate" answer of repair on behalf of the fibres of the injured muscle, they auto-block their activation before myoblasts are formed (Aarimaa et al., 2004). In the mature skeletal muscle there exists at least two principle populations of satellite cells (Rantenen et al., 1995; Jancowski et al., 2002; Kalimo et al., 1997; Qu-Peterson et al., 2002;; Rouger et al., 2004; Zammit et al., 2004). The "classic" satellite cells which reside under the basal lamina of the muscular fiber and which can be divided into"committed satellite cells" which are ready to differentiate themselves into myoblasts immediately after the harmful event and the "stem sateliite cells" which have to first undergo cellular division to be able to differentiate (Kalimo et al., 1997; Rantenen et al., 1995; Zammit et al.,2004). Through this cellular division (which may be seen form a biological point of view as a true and proper proliferation process), the population of stem satellite cells, again builds up the reserve of satellite cells for a future possible regenerative request (Rantenen et al., 1995; Zammit et al., 2004). In this population of satellite cells, we may note the existence of and under-population of cells capable of differentiation, beyond the myogenic lines, not only in mesenchymal lines but also in neural or endothelial ones (Jankowski et al., 2002; Qu-Peterson et al., 2002). Up until to today the satellite cells were retained the only resource of the myonucleus in the course of muscular repair (Chargé and Rudnicki, 2004), recent discovery has shown the presence of a different population of multi-powerful stem cells, which can contribute to the reparation of the injured skeletal muscle; the "non-muscle-resident stem cells" (Chargé and Rudnicki, 2004). In fact, even some isolated progenitor cells of the bone marrow (BM), the neuronal compart‐ ment, and different mesenchymal tissue, are able to differentiate in myogenic lines. The cells derived from BM, not only contribute to the regeneration of the muscles fibers in the injured skeletal muscle, but they are also able to re-integrate the pool of the satellite cells in the injured skeletal muscle (Labarge and Blau, 2002). In each case, it is important to note that the frequency at which these events happen seems to be very low, also in the case of serious lesion, if compared to the number of regenerated myoblasts derived from the "muscle-resident" satellite cells (Grounds et al., 2002; Labarge and Blau, 2002). So, it is quite disputable the fact that the "non muscle-resident" stem cells may give a significant contribution to the repair of injured skeletal muscle (Ground et al., 2002) In addition to the classic satellite cells, resident in the lower part of the basal lamina, there also exists a distinct population of extra-lamina collocated stem cells, inside the connective tissue of the skeletal muscle (Dreyfus et al., 2004). In answer to a harmful event on the skeletal muscle, these cells take part in the formation of

confirming this hypothesis, it is possible to note how the concentration of lactate inside the injured area, diminishes only slightly at the moment in which the concentration of O2 rises (Hunt et al., 1978). To this end, it is worth it to mention how some authors report values of lactate concentration, inside the injured muscle, between 8 and 18 mmol.1-1 (Hunt and Hussain, 1993). On the other hand, the hypothesis, that lactate was implied in the synthesis of collagen, it had already been put forward by some authors more than forty years ago (Green and Goldberg, 1963; Levine and Bates, 1976). In these experiments, it was described how lactate was implied in the synthesis of collagen, the authors noted how in their experiments the fibroblats put into culture, produced a higher quantity of collagen, in comparison with the control group when the concentration of lactate surpassed 20mmol.1-1. In these, just as in other successive experiments of such kind, it was observed in a hypoxic regime, the production of collagen is delayed up until the moment in which the hypoxic cells are not supplied with oxygen. In other words, the production of collagen only starts when there is the contextual presence of oxygen and lactate. This data suggests how the effect of lactate is independent in comparison to that of oxygen (Comstock and Udenfriend, 1970). However, in spite of the first stimulating results, this line of research has been practically abandoned since 1976. After nearly 20 years, other authors speculated that the lactate could work as a regulator in the process of collagen synthesis inside the injured tissue (Hunt and Hussain, 1993). According to these authors, the maximum ratio of collagen production, would occur in the presence of a high concentration of lactate, included between 8 and 18 mmol.1-1, concurrent with an high value of PO2, equal to about 100 mm Hg. This data, at a first look, would seem paradoxical, since we can usually consider logical a strong presence of lactate where there are scarce conditions of O2. However, we need to remember that the leucocytes are responsible for the production, in aerobic conditions, for an important quantity of lactate inside the injured tissue area, and that the production of lactate on behalf of the leucocytes inside the injury remains high also in the presence of a high value of PO2 (Levine and Bates, 1976). This type of biological model, characterized by a high concentration of contextual lactate to a high value of PO2, would establish a favorable condition, not only to the collagen synthesis but also to the angiogenesis (Hunt and Hussain, 1993) and in addition it is also probable that the lactate serves as stimulus

34 Muscle Injuries in Sport Medicine

for the secretion of TGF-β in the injured area (Falanga et al., 1991).

**23. The repair and re-modeling phase of the muscular fibers**

Once the phase of destruction has dropped in intensity, the real repair process of the injured muscle begins, which shows itself through two concurrent processes, which show between themselves, at the same time, complementary and antagonistic: the repair of the destroyed myofibers and of their respective innervations and the formation of healing tissue. A balanced progression of these two processes, makes up an essential pre-requisite for an optimal reactivation of the contractile functions of the skeletal muscle (Kalimo et al., 1997; Hurme and Kalimo, 1991;). In spite of the fact that the muscular fibers are, in general, considered as fibers of irreversibly post-mitotic type, the notable repair potential of the skeletal muscle is guaran‐ teed by an innate mechanism able to reactivate the injured contractile apparatus. Consequent‐

myoblasts and in the differentiation of myotubes (Chargé and Rudnicki, 2004). After the cylinders of the old basal lamina have been filled with new myofibers, the myofiber itself extends, through the opening of the basal lamina, towards the healing connective tissue, which has been formed between the stumps of the survived myofibers (Hurme et al., 1991; Kalimo et al., 1997). On both parts of the scar of connective tissue, the myofibers and the stumps of the survived fibers, in the attempt to pass through the scar which separates them, form multiple branches (Hurme et al., 1991). After trying to extend, for a short distance, the branches start to adhere to the connective tissue with their final points, forming mini MTJs with the scar tissue. In time, the scar area progressively diminishes in dimension, conducting the stumps to join with each other (Vaittinen et al., 2002). Even so it is still not well known the stumps of the sheared fibers on the opposite parts of the scar tissue, fuse totally between themselves at the end of the regenerative process or if, on the contrary, there remains some form of septum of connective nature (Vaittinene et al., 2002; Aarima at al., 2004). It has also been amply shown how the repair capacity of the skeletal muscle, in answer to trauma, is significantly reduced in the course of life. (Järvinen et al., 1983). This drop in regenerative capacity is not apparently attributable to a drop in the number or of the activity of the satellite cells (Järvinen et al., 1983) but rather to a complex drop in repair capacity of the muscles in an elderly person, such as it seems that each phase of repair process slows down and deteriorates with age (Järvinen et al., 1983).

precocious elastic behavior on behalf of the neo-granulation tissue of the injured skeletal mus‐ cle (Järvinen et al., 2000; 2003a; 2003b). The expression of the fibronectin is later followed by the type III collagen (Lehto et al., 1985; 1986; Goetsch et al., 2003; Hurem et al., 1991; Best et al., 2001; Ground et al., 2002), the production of type I collagen, on the contrary, only starts a couple of days later, to then remain elevated for several weeks (Lehto et al., 1985a; 1985b; Hurme et al., 1991; Best et al., 2001; Yan et al., 2003). The initial ample granulation tissue (i.e. the scar which forms between the stumps of the injured fibers ) concentrates a high degree of mechanical effi‐ ciency in a particularly reduced area of connective tissue, an area which is mainly composed of type I collagen (Järvinen, 1975; Lehto et al., 1985a; 1985b; Hurme et al., 1991; Järvinen and Leh‐ to, 1993). Despite the diffused preconception that the formation of fibrosis makes up an inevita‐ ble process in the natural history of muscular damage (Huard et al., 2002), the increase in the connective intramuscular tissue, in effect, does not increase in a substantial manner in an in‐ jured muscle, unless the muscle itself is not completely immobilized for an excessive length of time (Järvinen 1975; Lehto et al., 1985a; Järvinen and Lehto, 1993). The connective tissue scar, which is formed in the injury area represents the weak point of the injured muscle in the imme‐ diate post-traumatic phases (Hurme et al., 1991; Kääriäinen et al., 1998); however, its capacity of tensile force, increases considerably with the production of type I collagen (Lehto et al., 1985a ; 1985b; Kääriäinen et al., 1998). The mechanical stability of collagen, in its turn, is due to the formation of intermolecular cross-links, which form during the maturity of the scar tissue (Lehto et al., 1985b). Approximately ten days after the trauma, the maturity of the scar has reached a phase in which it no longer represents the structurally weak ring of the chain inside the injured muscle, so that, if the latter is stretched until break point, the damage generally oc‐ curs inside the adjacent tissue rather than where new mini MTJs have been formed between the repaired myofibers and the scar tissue (Järvinen, 1975; Järvinen, 1976; Kääriäinen et al., 1998). In any case, it will still need a long period of time before the strength of the muscle has com‐ pletely recovered. (Järvinen, 1975; 1976; Kääriäinen et al., 1998). Even though a large part of harmful events on the skeletal muscle heals without the formation of a debilitating fibrotic scar from a functional point of view, the proliferation of fibroblasts can be excessive and hesitate in the formation of thick scar tissue inside the injured muscle. In these cases, which are generally associated with superior levels of muscular trauma, and above all to those which are recurring, the scar can create a mechanical barrier which delays, or sometimes strongly limits, the repair of the myofibers through the gap formed by the damage (Järvinen, 1975; 1976). Some of these experimental studies have recently given interesting clarification regarding the scar formation in the injured skeletal muscle; we have been able to ascertain in fact, how direct application is of a particular form of small leucine-rich proteoglycan (SLRP), of decorin and of an antifibrotic agent like suramin or the γ-interferon, are able to inhibit the scar formation in the injured skele‐ tal muscle (Fukushima et al., 2001; Chan et al., 2003; Foster et al., 2003). The decorin, the sura‐ min and the γ-interferon are all specific inhibitors of the TGF-β (Yamaguchi et al., 1990; Grounds, 1991; Hildebrand et al., 1994; Chan and Foster, 2003) a growth factor which is held re‐ sponsible for the scar formation during the repair processes of the muscle. In addition to the in‐ hibiting action towards the TGF-β, the decorin and the SLRP, even though they can't tie themselves to the different collagens, are however able to regulate the fibrillogenesis and the assembly of the type I collagen fibrils (Frank et al., 1999; Nakumura et al., 2000; Corsi et al.,

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2002).

### **24. The formation of connective scar tissue**

Immediately after a muscular trauma, the gap formed in correspondence with the fibers, is fil‐ led with a hematoma, within the first day the pro-inflammatory cells, including the phago‐ cytes, invade the hematoma itself and start to form blood coagulation (Hurme et al., 1991b; Tidball, 1995; Cannon and Pierre, 1998). The fibrin and the fibronectin tie together to form both an initial granulation tissue and an initial ECM, which will serve as a scaffold and as an anchor‐ age site for the successive invasion on behalf of the fibroblasts (Hurme and Kalimo, 1991). We need to remember, to this end, how some fibroblasts present in the granulation tissue, may also derive from the myogenic cells (Li and Huard, 2002). It is very important to underline the fact that this neo-formed tissue bestows upon the tissue of the injured area the initial resistance to be able to support the contraction forces applied to the latter (Lehto et al.,1985; 1986; Hurme et al., 1991c). Following this, the fibroblasts start the synthesis of the proteins and of the proteo‐ glycans of the ECM, to restore the integrity of the framework of the connective tissue (Lehto et al., 1985; 1986; Hurme et al., 1991c; Goetsch et al., 2003). Amongst the first synthesized proteins of the ECM, there is tenacin- C (TN-C) and fibronectin (Lehto et al., 1986; Hurme and Kalimo, 1991; 1992; Goetsch et al., 2003) which initially change direction in multimeric fibrils to then form super-fibronectin, a protein which has much better adhesive properties (Morla et al., 1994; Wierzbika- Patynowski and Schwarzbauer, 2003). Both the fibronectin and the TN-C, due to their elastic properties, are able to support a remarkable number of elongation cycles, in respect to their rest length. These elongations, which are due to mechanical loads applied on a tissue level, play an important role both in the production of force and for the apparition of the first precocious elastic behavior on behalf of the neo-granulation tissue of the injured skeletal mus‐ cle (Järvinen et al., 2000; 2003a; 2003b). The expression of the fibronectin is later followed by the type III collagen (Lehto et al., 1985; 1986; Goetsch et al., 2003; Hurem et al., 1991; Best et al., 2001; Ground et al., 2002), the production of type I collagen, on the contrary, only starts a couple of days later, to then remain elevated for several weeks (Lehto et al., 1985a; 1985b; Hurme et al., 1991; Best et al., 2001; Yan et al., 2003). The initial ample granulation tissue (i.e. the scar which forms between the stumps of the injured fibers ) concentrates a high degree of mechanical effi‐ ciency in a particularly reduced area of connective tissue, an area which is mainly composed of type I collagen (Järvinen, 1975; Lehto et al., 1985a; 1985b; Hurme et al., 1991; Järvinen and Leh‐ to, 1993). Despite the diffused preconception that the formation of fibrosis makes up an inevita‐ ble process in the natural history of muscular damage (Huard et al., 2002), the increase in the connective intramuscular tissue, in effect, does not increase in a substantial manner in an in‐ jured muscle, unless the muscle itself is not completely immobilized for an excessive length of time (Järvinen 1975; Lehto et al., 1985a; Järvinen and Lehto, 1993). The connective tissue scar, which is formed in the injury area represents the weak point of the injured muscle in the imme‐ diate post-traumatic phases (Hurme et al., 1991; Kääriäinen et al., 1998); however, its capacity of tensile force, increases considerably with the production of type I collagen (Lehto et al., 1985a ; 1985b; Kääriäinen et al., 1998). The mechanical stability of collagen, in its turn, is due to the formation of intermolecular cross-links, which form during the maturity of the scar tissue (Lehto et al., 1985b). Approximately ten days after the trauma, the maturity of the scar has reached a phase in which it no longer represents the structurally weak ring of the chain inside the injured muscle, so that, if the latter is stretched until break point, the damage generally oc‐ curs inside the adjacent tissue rather than where new mini MTJs have been formed between the repaired myofibers and the scar tissue (Järvinen, 1975; Järvinen, 1976; Kääriäinen et al., 1998). In any case, it will still need a long period of time before the strength of the muscle has com‐ pletely recovered. (Järvinen, 1975; 1976; Kääriäinen et al., 1998). Even though a large part of harmful events on the skeletal muscle heals without the formation of a debilitating fibrotic scar from a functional point of view, the proliferation of fibroblasts can be excessive and hesitate in the formation of thick scar tissue inside the injured muscle. In these cases, which are generally associated with superior levels of muscular trauma, and above all to those which are recurring, the scar can create a mechanical barrier which delays, or sometimes strongly limits, the repair of the myofibers through the gap formed by the damage (Järvinen, 1975; 1976). Some of these experimental studies have recently given interesting clarification regarding the scar formation in the injured skeletal muscle; we have been able to ascertain in fact, how direct application is of a particular form of small leucine-rich proteoglycan (SLRP), of decorin and of an antifibrotic agent like suramin or the γ-interferon, are able to inhibit the scar formation in the injured skele‐ tal muscle (Fukushima et al., 2001; Chan et al., 2003; Foster et al., 2003). The decorin, the sura‐ min and the γ-interferon are all specific inhibitors of the TGF-β (Yamaguchi et al., 1990; Grounds, 1991; Hildebrand et al., 1994; Chan and Foster, 2003) a growth factor which is held re‐ sponsible for the scar formation during the repair processes of the muscle. In addition to the in‐ hibiting action towards the TGF-β, the decorin and the SLRP, even though they can't tie themselves to the different collagens, are however able to regulate the fibrillogenesis and the assembly of the type I collagen fibrils (Frank et al., 1999; Nakumura et al., 2000; Corsi et al., 2002).

myoblasts and in the differentiation of myotubes (Chargé and Rudnicki, 2004). After the cylinders of the old basal lamina have been filled with new myofibers, the myofiber itself extends, through the opening of the basal lamina, towards the healing connective tissue, which has been formed between the stumps of the survived myofibers (Hurme et al., 1991; Kalimo et al., 1997). On both parts of the scar of connective tissue, the myofibers and the stumps of the survived fibers, in the attempt to pass through the scar which separates them, form multiple branches (Hurme et al., 1991). After trying to extend, for a short distance, the branches start to adhere to the connective tissue with their final points, forming mini MTJs with the scar tissue. In time, the scar area progressively diminishes in dimension, conducting the stumps to join with each other (Vaittinen et al., 2002). Even so it is still not well known the stumps of the sheared fibers on the opposite parts of the scar tissue, fuse totally between themselves at the end of the regenerative process or if, on the contrary, there remains some form of septum of connective nature (Vaittinene et al., 2002; Aarima at al., 2004). It has also been amply shown how the repair capacity of the skeletal muscle, in answer to trauma, is significantly reduced in the course of life. (Järvinen et al., 1983). This drop in regenerative capacity is not apparently attributable to a drop in the number or of the activity of the satellite cells (Järvinen et al., 1983) but rather to a complex drop in repair capacity of the muscles in an elderly person, such as it seems that each phase of repair process slows down and deteriorates with age (Järvinen

Immediately after a muscular trauma, the gap formed in correspondence with the fibers, is fil‐ led with a hematoma, within the first day the pro-inflammatory cells, including the phago‐ cytes, invade the hematoma itself and start to form blood coagulation (Hurme et al., 1991b; Tidball, 1995; Cannon and Pierre, 1998). The fibrin and the fibronectin tie together to form both an initial granulation tissue and an initial ECM, which will serve as a scaffold and as an anchor‐ age site for the successive invasion on behalf of the fibroblasts (Hurme and Kalimo, 1991). We need to remember, to this end, how some fibroblasts present in the granulation tissue, may also derive from the myogenic cells (Li and Huard, 2002). It is very important to underline the fact that this neo-formed tissue bestows upon the tissue of the injured area the initial resistance to be able to support the contraction forces applied to the latter (Lehto et al.,1985; 1986; Hurme et al., 1991c). Following this, the fibroblasts start the synthesis of the proteins and of the proteo‐ glycans of the ECM, to restore the integrity of the framework of the connective tissue (Lehto et al., 1985; 1986; Hurme et al., 1991c; Goetsch et al., 2003). Amongst the first synthesized proteins of the ECM, there is tenacin- C (TN-C) and fibronectin (Lehto et al., 1986; Hurme and Kalimo, 1991; 1992; Goetsch et al., 2003) which initially change direction in multimeric fibrils to then form super-fibronectin, a protein which has much better adhesive properties (Morla et al., 1994; Wierzbika- Patynowski and Schwarzbauer, 2003). Both the fibronectin and the TN-C, due to their elastic properties, are able to support a remarkable number of elongation cycles, in respect to their rest length. These elongations, which are due to mechanical loads applied on a tissue level, play an important role both in the production of force and for the apparition of the first

et al., 1983).

36 Muscle Injuries in Sport Medicine

**24. The formation of connective scar tissue**
