| BackgroundMuscle injuries are one of the most common injuries occurring in sports, their frequency varying from 10-55% of all sustained injuries. Muscle injuies can result form a variety of mechanisms including contusion, strain and laceration, etc. More than 90% of the injuries are contusion or strain. The process of muscle repair follows a rather constant pattern irrespective of the different injury mechanisms. The healing process comprising three phases, including destruction phase, repair phase and remodelling phase. The number of adult skeletal muscle cells remained constant,the regenerative ability of skeletal muscle is largely dependent on muscle satellite cells. Muscle satellite cells are quiescent precursors interposed between an individual myofiber and the basal lamina. In addition muscle satellite cells are able to self-renew and to give rise to differentiated cells.Muscle injuries present a challenge in trauma rehabilitation. The healing process of muscle injury is very slow and often results in incomplete functional recovery. Besides, it is often hindered by the formation of scar tissue, which may contribute to the tendency for recurrent injuries. The current commonly used treatment methods include the local application of heat, immobilization and passive range of motion exercises,non-steroidal anti-inflammatory drugs (NSAIDs), intramuscular corticosteroids, and even surgery. But these therapies remain suboptimal.Ultrasound (US) therapy is convenient, inexpensive, safe and non-invasive, which is commonly used in clinical treatment of various types of tissue injuries. Therapeutic ultrasound has been used for treating muscle injuries during inflammatory, proliferative and remodeling phases. However, opinions still differ concerning the effect of ultrasound therapy on muscle injuries, and conflicting results on the efficacy of therapeutic ultrasound in treating muscle injuries have been reported.Due to lack of scientific theoretical basis, many of the decisions made regarding how to utilize therapeutic ultrasound in the treatment of contusion injuries to skeletal muscle are based on personal opinion and experience with the modality. ObjectiveThe purpose of this study was to examine the effects of pulsed ultrasound on several biomarkers of skeletal muscle regeneration and scar generation based on a standardized skeletal muscle contusion, and the effects of pulsed ultrasound on muscle biomechanical properties following contusion were studied too,so as to provide an objective basis for clinical applications of ultrasound therapy of muscle injury.Methods1. Twenty-four 3-month-old adult male SD rats received a bilateral contusion injury to the gastrocnemius via a weight-dropping technique,the injury was accomplished using an instrument developed by ourselves.The trauma was delivered by a falling object weighing 220g from a distance of 110cm onto the gastrocnemius muscle.The force delivered by a falling object with a constant 2.16N,and the falling object possesses 2.37J of kinetic energy as it strikes the muscle. The activity of creatine kinase of each rat was measured in 0-hour animals and 2, 24 hours after muscle injury used as an indicator of skeletal muscle injury.2. Forty-eight 3-month-old adult male SD rats received a reproducible contusion injury to the right gastrocnemius muscles by the same technique as above.The animals were randomly divided into four groups(n=12 each):(1)control group (muscle injury without treatment),(2) muscle injury and ultrasound therapy of 0.25W/cm2,(3) muscle injury and ultrasound therapy of 0.5W/cm2,(4) muscle injury and ultrasound therapy of 0.75W/cm2. At 24h post-injury,pulsed ultrasound (frequency, 3MHz) was used for groups 2,3, and 4,and delivered for 5 minutes daily on the right injury hindlimb . Four animals in each group were sacrificed at day 4, 7, and 14 post-injury.The speed of myoregeneration was evaluated by using immunohistochemical stainings for desmin and hematoxylin and eosin (HE).3. Forty-eight 3-month-old adult male SD rats received a reproducible contusion injury to the right gastrocnemius muscles by the same technique as above.The animals were randomly divided into two groups(n=24 each):(1)control group (muscle injury without treatment),(2)ultrasound group (muscle injury followed by ultrasound therapy).At 24h post-injury pulsed ultrasound treatment (frequency, 3MHz;intensity,0.25W/cm2) was used for group 2, and delivered for 5 minutes daily on the right damaged hindlimb.The animals were sacrificed at 2,4, 7, and 14 days post-injury,six treated and six control animals were sacrificed each time. The speed of myoregeneration and fibration were evaluated by using immunohistochemical stainings for actin, collagen I and collagenâ…¢, and hematoxylin and eosin (HE) staining.4. Thirty 3-month-old adult male SD rats. Twenty-four of the 30 animals received a bilateral contusion injury to the gastrocnemius by the same technique as above,the remaining six were normal animals.Pulsed ultrasound treatment (frequency, 3MHz; intensity,0.25W/cm2) was started 24h post-injury and delivered for 5 minutes daily. Treatment was on the left hindlimb and the contralateral right hindlimb was the nonultrasound control. At 14, 21,28,and 35 days after injury, the biomechanical performance of the injured gastrocnemius were tested and then compared with their own control and normal group.Results1. In this experiment, the results showed that the peak of the serum CK activity of the rats occurred at 2 hours after injury,but it declined rapidly on 24h post-injury. The serum CK activitives were significantly increased in 2, 24 hours animals post-injury when compared to the uninjured 0-hour animals respectively(P<0.05). Serum CK levels were increased in muscle injured animals, indicating that the contusion animal model was successful.2. In this experiment, the desmin-positive satellite cells were significantly more numer- ous in the groups 2,3, and 4 at day 4,7 and 14 compared with control group(P<0.05),but there were no statistical differences among the 3 ultrasound groups(P>0.05).3. In this experiment, the actin-positive satellite cells were significantly more numerous in ultrasound group at day 4,7 and 14 compared with control group(P<0.05). But the type I collagen expression of control group was significantly higher than that of the ultrasound group at day 7 and 14(P<0.05), the typeâ…¢collagen expression of control group was significantly higher than that of the ultrasound group at day 4,7 and 14(P<0.05).4. In this experiment, we found it has shown an increase in biomechanical properties after ultrasound therapy.The limit load and yield strength of ultrasound group have been restored to normal value at day 28(P>0.05), and the spontaneous recovery group was still lower than normal group at day 35 (P<0.05).The wet weight and tensile strength of ultrasound group have been restored to normal value at day 35(P>0.05),but the spontaneous recovery group was still lower than normal group (P<0.05).ConclusionsThe contusion animal model was established successful via a weight-dropping technique,the injury was accomplished using an instrument developed by ourselves.Skeletal muscle has an impressive ability to regenerate in response to injury, the regenerative ability of skeletal muscle is largely dependent on muscle satellite cells. Muscle satellite cells proliferation were enhanced significantly by the pulsed ultrasound therapy during the stage of regeneration ,and also have a considerable effect on prevention of muscle fibrosis and improvement in muscle biomechanical properties.There have no significant dose-dependency effects among the intensity range of 0.25-0.75W/cm2. The pulsed ultrasound plays a beneficial role in the skeletal muscle healing process following contusion injury and improve the quality of injured skeletal muscle.
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