Plasticity of allogenic muscle tissue transplanted into a traumatized rat muscle subjected He-Ne laser radiation

The effect of muscle tissue alloplasty and laser radiation on the post-traumatic regeneration of skeletal muscles was studied. The implantation of an unirradiated minced allogenic muscle tissue into the area of trauma of a previously laser-irradiated muscle had a positive effect. The allogenic muscle tissue underwent active regeneration. The traumatized muscle fibers in the rat-recipient muscle grew into the alloplant. The implantation of a previously laser-irradiated muscle tissue into the area of trauma of an unirradiated muscle was less effective. The allogenic muscle tissue prevented the growth of rat-recipient myofibers and underwent lysis in general. The allotransplantation of a laser-irradiated muscle tissue into an x-irradiated (20 Gy) traumatized muscle increased the destruction of rat-recipient muscle tissue.     

Regeneration of the x-ray irradiated gastrocnemius muscle in old rats after stimulation

In the present study the implantation of nonexposed muscle tissue to the site of injury in irradiated musculus gastrocnemius and following laser therapy were applied in order to stimulate this muscle's posttraumatic regeneration in old rats. It was shown that a far larger amount of functionally active muscle tissue was formed at the site of injury compared with rats received laser therapy alone or the ones which were only implanted nonexposed minced muscle tissue. The muscle tissue consisted of muscle fibers which originated from the grafted pieces of nonexposed skeletal muscle and the ones produced by myofibers of muscle stumps recovered after irradiation. The connective tissue developed more evenly. The formation of adipose tissue was not observed at the site of injury. Moreover, the skin wound healing and the hair growth were stimulated as well.     

Multiple regeneration from axolotl limb stumps bearing cross-transplanted minced muscle regenerates.

Flexor and extensor muscles in the upper arms of axolotls were minced and cross-transplanted. The limbs were amputated 5 and 30 days after mincing. In each experiment a high percentage of the regenerates consisted of multiple limbs. This demonstrates that the morphogenetic information which produces multiple regenerates after the cross-transplantation of limb stump muscle is stable enough to be expressed after both mincing and the subsequent tissue regeneration of the muscle.     

Effect of helium-neon laser rays on the processes of postradiation recovery in the skeletal muscles of old rats

The present experiments were conducted to determine the stimulant effect of helium-neon laser on the postradiation recovery in irradiated uninjured skeletal muscle of rats aged 2-2.5. This was indicated by a restored ability of the muscle for posttraumatic regeneration. The both hind rat legs were exposed to local irradiation of 20 Gy and following laser therapy (8-9 procedures at 3 min each, impulsive or continuous one). Then both musculus gastrocnemius were cut across 30 days after irradiation. It was shown that laser therapy employed before injury of the irradiated muscle accelerated fibrin resorption and improved connective tissue, but slightly stimulated muscular tissue. Impulse laser therapy was more favourable for state of skin and healing of the skin wound after irradiation.     

Tissue interactions of regenerating muscle tissue with skeletal muscle under the effect of ionizing radiation

The effect of the muscle tissue, ground into a fine chyme, to restoration of the m. gastrocnemius ability to posttraumatic regeneration after x-ray radiation in the doses 20--30 Gy, has been studied in 64 non-inbred white male rats with the body mass about 200 g. The ground muscle is a viscous mass, consisting of finest scraps of muscle fibers, pieces of sarcolemmal tubules with 2--3 nuclei and separate cells. The great destruction of the muscle stroma relieves metabolites to get out and to diffuse. As demonstrate the histological investigations carried out for 1 month, an essential part of the muscle autotransplant, put into the irradiated extremity, regenerates up to formation of differentiated muscle fibers and promotes to restore the regenerative activity in the irradiated m. gastrocnemius.     

Plasticity of allogeneic muscle tissue implanted into a traumatized rat muscle on exposure to He-Ne laser light

The combined effect of muscle tissue alloplasty and He-Ne laser light on the regeneration of a traumatized skeletal muscle was studied. In implantation of untreated allogeneic tissue into the region of transverse cutting of the gastrocnemius muscle exposed to laser light in vivo before surgery, the muscle regeneration in recipient rats was successful. Active regeneration of the allogeneic tissue and the muscle tissue of the recipient rat was noted. In implantation of laser-exposed allogeneic tissue into the region of trauma of unexposed gastrocnemius muscle, the muscle regeneration was less successful. The alloplastic implant was replaced by connective tissue and prevented the growth of the muscle tissue of the recipient rat in the defective zone. Implantation of laser-exposed allogeneic muscle tissue into the region of trauma of the muscle x-irradiated to a dose of 20 Gy enhanced destructive processes in it.     

A monoclonal antibody stains myogenic cells in regenerating newt muscle

Monoclonal antibodies have been used to study minced muscle regeneration in the adult newt, Notophthalmus viridescens. The contralateral limb was amputated and the immunostaining patterns in the regenerating blastema were compared with the minced tissue in sectioned material. Staining with a myofibre-specific antibody, called 12/101 (Kintner & Brockes, 1984), showed that myofibre degeneration was complete by 8-10 days after mincing, with myogenesis commencing 2 days later. Another monoclonal antibody, called 22/18, previously shown to label a subset of cells in the regeneration blastema of the newt (Kintner & Brockes, 1984, 1985), was found also to recognize a population of cells in regenerating minced muscle. At 6 days after mincing, the number of 22/18-positive (22/18+) cells was low but by days 12-16, during the period of myogenesis, their number had increased to become a major population within the minced tissue. A small number of the 22/18+ cells could be double labelled with 12/101 at this time. Prior to this, there was a phase in which 12/101 staining had disappeared from the mince. Cells immunoreactive with both antibodies after this phase confirm that at least some of the 22/18+ cells are myogenic. The number of 22/18+ cells decreased as muscle repair and maturation progressed. These results show that 22/18 is not specifically associated with blastemal cells but is a more general marker for regenerating systems in the newt. They further suggest an alternative interpretation of the double-labelled cells used by Kintner & Brockes (1984) as evidence for myofibre dedifferentiation in limb regeneration. Instead, we propose that such cells represent new myogenesis occurring by tissue repair of locally damaged muscle fibres.     

Transplantation of devitalized muscle scaffolds is insufficient for appreciable de novo muscle fiber regeneration after volumetric muscle loss injury

Volumetric muscle loss (VML) is a traumatic and functionally debilitating muscle injury with limited treatment options. Developmental regenerative therapies for the repair of VML typically comprise an ECM scaffold. In this study, we tested if the complete reliance on host cell migration to a devitalized muscle scaffold without myogenic cells is sufficient for de novo muscle fiber regeneration. Devitalized (muscle ECM with no living cells) and, as a positive control, vital minced muscle grafts were transplanted to a VML defect in the tibialis anterior muscle of Lewis rats. Eight weeks post-injury, devitalized grafts did not appreciably promote de novo muscle fiber regeneration within the defect area, and instead remodeled into a fibrotic tissue mass. In contrast, transplantation of vital minced muscle grafts promoted de novo muscle fiber regeneration. Notably, pax7+ cells were absent in remote regions of the defect site repaired with devitalized scaffolds. At 2 weeks post-injury, the devitalized grafts were unable to promote an anti-inflammatory phenotype, while vital grafts appeared to progress to a pro-regenerative inflammatory response. The putative macrophage phenotypes observed in vivo were supported in vitro, in which soluble factors released from vital grafts promoted an M2-like macrophage polarization, whereas devitalized grafts failed to do so. These observations indicate that although the remaining muscle mass serves as a source of myogenic cells in close proximity to the defect site, a devitalized scaffold without myogenic cells is inadequate to appreciably promote de novo muscle fiber regeneration throughout the VML defect.     

Isoenzyme studies of whole muscle grafts and movement of muscle precursor cells

Summary Isoenzymes of glucose-6-phosphate isomerase (GPI: E.C. 5.3.1.9) were used as markers to determine the origin of cells which give rise to new muscle formed in allografts of whole intact muscle. GPI isoenzymes were also employed to see whether host precursor cells, which have been shown to contribute to muscle formation in grafts of minced muscle, can be derived from muscle lying adjacent to grafts.Excellent muscle regeneration was found in allografts of extensor digitorum longus (EDL) muscle examined after 58 days: 12 of 16 grafts contained 80% or more new muscle. Isoenzyme analysis showed that most, and in 2 instances all, new muscle was derived from implanted donor cells; however, there was strong evidence that in 5 grafts some, or all, new muscle must have resulted from host cells moving into the graft. Although hybrid isoenzyme was not detected this was attributed to factors associated with host tolerance which appear to interfere with fusion between host and donor myoblasts.Isografts of minced muscle were placed next to whole EDL muscle allografts to see if cells from allografts moved into adjacent regenerating tissue. Unfortunately, muscle regeneration in minced isografts was poor; only 3 contained 50% or more new muscle and most contained large amounts of fibrous connective tissue. Only a single isoenzyme band was detected in 11 isografts, but in five instances, the presence of a second band showed that cells from EDL allografts were also present. As no hybrid isoenzyme was detected, it is not known whether these cells which had moved into the regenerating minced grafts were muscle precursors, fibroblasts or some other cell types.     

Muscle regeneration and the state of the thymus in adult rats under laser irradiation and alloplasty of newborn gastrocnemius muscle and diaphragm

The regeneration of adult rat gastrocnemius muscles has been studied under implantation of new-born rat muscles into area of muscle trauma. Alloplasty was performed using minced gastrocnemius and diaphragm muscles, which differs at birth in animals by degree of differentiation. The rat-recipient area of alloplasty was subjected to He-Ne laser radiation before operation, with the aim of reducing the immune response to allogenic muscle tissue. It has been shown that the number of regenerating myofibers produced from implanted gastrocnemius muscles is more than in alloplants from diaphragms. However, the formation of cartilage, bone, and adipose tissue foci was observed in the alloplastic region throughout the whole regeneration period. After implantation of minced diaphragm muscles, cartilage nodes were observed only in 7-day regenerates. At the end of observation, in the first instance, the area of muscle trauma in adult rat muscles was replaced by adipose tissue, even in the case of initial laser irradiation. After the implantation of diaphragm muscles, the area of trauma was filled with regenerating muscle tissue.     

Morphofunctional characteristics of the thymus and muscle regenerates under laser irradiation and alloplasty of adult muscle tissue in the area of trauma

The effect of muscle tissue alloplasty and He-Ne laser radiation on the skeletal muscle regeneration and thymus function was studied. Allogenic muscle tissue was implanted from an adult rat. Without laser irradiation (series 1), initial enhancement of thymus recovery observed on day 7 after alloplasty (a characteristic stress response to operation) was followed by gradual destructive changes in the thymus tissue. On day 30 after alloplasty, connective tissue developed in the implantation area in muscle regenerates, and the muscle tissue accounted for 64 ± 2%. Implantation of unirradiated allografts into the muscles of recipient rats preirradiated with a He-Ne laser (series 2) resulted in a nearly complete destruction of the thymus and suppression of its function; the mitotic index of thymocytes was low. These changes were observed throughout the experiment starting immediately after the operation. In this case, the allogenic transplant retained the ability to develop: the 30-day repairing muscles consisted of 71 ± 2% of muscle tissue. When an allograft preirradiated with a He-Ne laser was implanted into unirradiated rats (series 3), thymus destruction at the beginning of the postoperative period was much less significant than in series 2 but more pronounced than in series 1. Then, thymus recovered more rapidly, the allogenic transplant was resorbed, and the muscle tissue in the regenerates accounted for 62 ± 3%.     

Functional analysis of limb recovery following autograft treatment of volumetric muscle loss in the quadriceps femoris

Severe injuries to the extremities often result in muscle trauma and, in some cases, significant volumetric muscle loss (VML). These injuries continue to be challenging to treat, with few available clinical options, a high rate of complications, and often persistent loss of limb function. To facilitate the testing of regenerative strategies for skeletal muscle, we developed a novel quadriceps VML model in the rat, specifically addressing functional recovery of the limb. Our outcome measures included muscle contractility measurements to assess muscle function and gait analysis for evaluation of overall limb function. We also investigated treatment with muscle autografts, whole or minced, to promote regeneration of the defect area. Our defect model resulted in a loss of muscle function, with injured legs generating less than 55% of muscle strength from the contralateral uninjured control legs, even at 4 weeks post-injury. The autograft treatments did not result in significant recovery of muscle function. Measures of static and dynamic gait were significantly decreased in the untreated, empty defect group, indicating a decrease in limb function. Histological sections of the affected muscles showed extensive fibrosis, suggesting that this scarring of the muscle may be in part the cause of the loss of muscle function in this VML model. Taken together, these data are consistent with clinical findings of reduced muscle function in large VML injuries. This new model with quantitative functional outcome measures offers a platform on which to evaluate treatment strategies designed to regenerate muscle tissue volume and restore limb function.     

Regenerative activity of muscle tissue and thymus status in young and old mdx mice with muscle injury and wound xenoplasty

Responses of the skeletal muscle tissue and thymus to muscle injury (complete transection) and wound xenoplasty with the minced muscle tissue of newborn rats (tissue therapy) were studied in mdx mice aged 12–16 and 40–48 weeks. The muscle tissue of mdx mice has genetic defects causing chronic dystrophic processes in it. The muscle tissue of young mdx mice proved to retain a relatively high capacity for regeneration. Under conditions of tissue therapy of the wound, the formation of muscle fibers from muscle cells of the graft and active regeneration of muscle fibers in the recipient mice were observed, and no structural defects were detected in the thymus. The capacity of posttraumatic regeneration in old mdx mice was lower. The xenogenic graft was undergoing resorption, thereby suppressing regeneration of muscle fibers and causing further tissue destruction in the injured muscle. The thymus parenchyma was subject to degenerative changes such as the formation of gaps, hemorrhagic foci, and increased numbers of macrophages and mast cells.     

Structural properties of regenerating muscle and the state of thymus after laser therapy of injured muscle in different periods of regeneration

The effect of He-Ne laser (632.8 nm, 2.5–3.0 mW/cm²) on skeletal muscle regeneration proved to depend on the period of regenerating tissue exposure to the radiation. Ten 3-min exposures within 1–15 days after muscle injury at a dose of 4.5–5.4 J/cm² per each operated limb accelerated the inflammation and regeneration of the muscle. The regenerates demonstrated a more muscular structure compared to laser therapy at later periods. The load on the thymus increased, its weight and cortical layer restoration decelerated, and aplasia was observed. The same dose of laser radiation in the period of days 16–30 increased the sclerotization of regenerating muscle tissue. The reactive changes in the thymus were less pronounced, the cortical layer recovery accelerated, and the mitotic index of thymocytes considerably increased. A three times lower laser radiation dose (1.5–1.8 J/cm²) in early regeneration inhibited inflammation and growth of the connective and muscle tissues compared to control. No increase in the functional activity of thymus was observed.     

Regenerative Capacity of Muscle Tissue and Thymus State in γ-Irradiated Rats Exposed to Long-term He–Ne Laser Radiation

We studied the effect of He–Ne laser on regeneration of damaged gastrocnemius muscle in rats irradiated at 6 Gy in conditions of fractional laser energy spread (10 exposures, 3 min for each limb, within 30 days after the operation; 2–3 exposures weekly; 2.5–3.0 mW/cm² power density; and 9.0–10.8 J/cm² total dose per animal). Laser radiation stimulated regenerative activity of the skeletal muscle and favored a more even distribution of load on the thymus (a smooth decrease in its weight and slow aplasia). The level of chromosomal aberrations in the thymocytes demonstrated certain instability although remained lower as compared to the control during the whole observation period (60 days).     

Reaction of intact skeletal muscle on the transplantation of minced muscle tissue

The experiments on rats have revealed that grafting of minced muscle tissue to unaffected gastrochemical muscle of the same animal leads to active regeneration in the graft accompanied by the formation of myogenic elements. In the zone of unaffected muscle contact with the graft the plastic state of muscle tissue was observed: sarcoplasm basophily of muscle fibers, groups and chains of round nuclei in them, the presence of satellite cells, ultrastructural changes, indicative of metabolic activity of the muscle fibers. With the resorption of the graft, these phenomena in the unaffected muscle gradually disappeared.     

Structure and functions of muscle allotransplants developing in adrenals and the alloplasty area after long-term exposure

The cross allotransplantation of right gastrocnemius muscles was carried out between intact rats and rats in which adrenals and right shins were subjected to low-energy He-Ne laser radiation (10 procedures for 2 weeks, the exposure duration 5 min; total dose for each rat 15-18 J/cm2). By conditions of experiment, the rats in each series were both donors and recipients, and hence the heterogeneity of rats in each series was the same. It was shown that the laser radiation in the dose studied and the regimes of exposure of rat adrenals and shin (area of the planned allotransplantation) influenced the muscle alloplasty. In rats with irradiated adrenals and shin, the disintegration and resorption of muscle allografts occurred more actively. In intact rats, muscle allografts were more viable. The allogenic muscle tissue and a weak contractile reaction of allografts were revealed in most of them. The allografts showed a moderate lymphocytic infiltration. The data obtained indirectly evidence that the transplant immune reaction increased in rats with irradiated adrenals and shin. In intact rats, the transplant immune reaction to irradiated m.gastrocnemius from irradiated shin decreased.     

The microscopic morphology of orthotopic free muscle grafts in rabbits

The flexor digitorum superficialis muscle was free grafted (without neurovascular anastomoses) in 122 rabbit forelimbs. Histologic nd histochemical examinations through 6 months after grafting were performed. An early ischemic necrosis of the entire graft, except for a few percent of fibers at the very surface, was consistently seen. Subsequently, there occurred a regeneration of muscle with reconstitution of up to 100 percent of normal numbers of fibers. There was a wide variation in the numbers of fibers regenerated; however, the fiber-free areas were then being replaced by connective tissue. Muscle grafts in 1-month-old rabbits regenerated faster and yielded muscle with evidence of more extensive reinnervation and less connective tissue than 3-month-old animals. In the early postgraft period, minced grafts appeared to be as good as whole ones, but after 1 month, they developed far more connective tissue. Differentiation into fast-twitch and slow-twitch muscle fibers and into high- and low-oxidative fibers began at 2 to 3 weeks after grafting but was not extensive until 3 months. At 6 months, grafts showed areas of normal-appearing muscle interspersed with areas that lacked signs of reinnervation. The earliest sign of regeneration is the appearance of several very elongated nuclei encircling each previously anucleate necrotic muscle fiber. A small amount of basophilic cytoplasm then appears around each new nucleus. As blood vessels grow into the graft, a centripetal wave of phagocytosis is seen, taking 2 to 3 weeks and leaving a bed of immature muscle fibers. We believe this to be the first documentation of regeneration's commencing prior to and thus independently of phagocytosis.     

Histological and histochemical studies on the nervous influence on minced muscle regeneration of triceps surae of the rat.

The degree of minced rat muscle regeneration in the absence of nerve fibers was compared with that of normal regenerates between one and 270 days postoperatively. Up to around 30 days, the number of muscle fibers and their morphology were comparable in both normal innervated and denervated regenerates; both showed clear cross striations and peripherally located nuclei. Histochemically, SDH and myofibrillar ATPase (pH=9.4) reactions were positive, but there were no typical signs of fiber types in either case of regeneration. The only consistent difference in the early period was the smaller fiber cross sectional areas in denervated regenerates than in innervated ones. Starting about 40 days, the muscle fibers in innervated regenerates became differentiated into different fiber types (fast-twitch-oxidative-glycolytic, FOG., fast-twitch-glycolytic, FG., slow-twitch-oxidative, SO.) but there were no such activities in denervated regenerates, although their SDH and myofibrillar ATPase reactions remained positive for a long time. Degenerating muscle fibers could no longer be identified in innervated regenerates. In the denervated regenerates, however, muscle fibers underwent atrophic or degenerative changes and were replaced by connective tissue. The complete disappearance of muscle fibers varied with individual regenerates. In some cases, it occurred about 90 days and in others, traces of muscle fibers could still be seen as late as 150 days postoperatively. Thus, nerves seem to be important primarily in the late phase of regeneration; namely, differentiation of fiber types and maintenance of the structural integrity of muscle fibers.     

Effects of helium-neon laser on regeneration capacity of skeletal muscles of adult guinea pigs]

The muscle regeneration was studied in across-cut gastrocnemius muscle of adult guinea pigs. Both hind guinea pig legs were exposed to laser therapy (3-9 procedures for 5 min each) after operation. It was shown that laser therapy accelerated fibrin resorption and wound healing, decreased the muscle fiber degeneration, increased DNA and RNA synthesis in regenerating tissues, stimulated regenerative muscle fiber capacity. However, the regenerating muscle tissue did not connect both muscle stumps, and the narrow connective tissue scar was formed. Before it was shown that the same laser therapy conditions were more effective for rats. Probably, the stimulating effect of laser rays on regenerative tissue ability depends on species peculiarities of animals.