New Approaches for the Treatment of Muscle Injuries

Dissertation / Doktorarbeit von Tamsin Wright Carpenter

Abstract:

Muscle injuries constitute up to 55% of all injuries sustained in sport events. The incidence and severity of these injuries is certainly greater in some sport modalities than in others. Significant morbidity, such as early functional and structural deficits, reinjury, atrophy, contracture, and pain, often occurs following muscle injuries, leading to loss of training and competition time. Healing of these injuries is a complex phenomenon depending of multiple factors, which are both within and outside the control of the clinician. On the whole, the best treatment regime has not yet been clearly defined, and the recommended treatment regimens have varied widely, depending on the severity of the injury.

Injuries to skeletal muscle during sport can occur by different mechanisms including blunt trauma in the case of contusions or stretch-induced injury in muscle strains. Another type of injury are those induced by laceration to the muscle but these are not particularly relevant in sport. A further complication of muscle injuries is the compartment syndrome, which generally occurs when tissues within an osteofascial compartment are compromised by increased pressure within the compartment.

Muscle strain may be a consequence of eccentric exercise, when the muscle develops tension during this type of lengthening contraction. These injuries are especially common in high-velocity situations in sports that require sprinting or jumping such as basketball, American football, rugby or soccer. The most susceptible muscles are the biarticular muscles such as the rectus femoris, the hamstrings and the gastrocnemius.

Interestingly, a high percentage of type II fibers or fast twitch fibers has been attributed to make muscles susceptible to strains because of their ability to contract fast and produce high forces. Furthermore, most muscle strains occur at or very near to the myotendinous junction (MTJ) of the superficial muscles working across two joints. In the worst case, muscle stretch-induced injuries can lead to muscle ruptures or tears. Jarvinen et al. have classified muscle strains into three categories according to their severity: Mild (first degree) strains where a few muscle fibers are torn, with minor swelling and discomfort but no or only minimal loss of strength and restriction of movements; Moderate (second degree) strains where there is a greater damage of the muscle with detection of a bleeding in the MRI scan and a moderate but not complete loss of strength; severe (third degree) strain in which we find a tear extending across the whole or most of the muscle belly, resulting in total loss of muscle function.

In contrast to muscle contusions, the haematoma can collect between the muscle tissue and the surrounding fascial compartment as shown by ultrasound. The time to recovery after muscle strain depends naturally on the severity of the injury as classified above. For example, after a hamstring strain, returning a patient to sport without the risk of suffering from reinjury can take from two weeks to six months.

Three different studies have been performed to investigate the effects of new approaches to improve the healing of muscle injuries: (1) Mice have been subjected to a muscle contusion and blood was collected to prepare an autologous conditioned serum (ACS) enriched with a variety of growth factors that was repeatedly injected to the site of injury; the time course of regeneration has been evaluated morphologically with special emphasis on the activation of satellite cells. (2) Treatment with ACS in comparison to conventional treatment was monitored in sportsmen with a 2nd degree muscle strain as controlled by MRI. (3) Electrotransfer of the FGF-2 gene was performed in mice with a contusion and muscle regeneration was monitored as in the first study. Administration of ACS as well as electrogene transfer substantially improved muscle healing to a similar extent; this result was determined by an increased and earlier activation of the satellite cells, allowing the regenerating muscle to return to full structural integrity earlier. The sportsmen treated with ACS showed an earlier recovery from injury than their conventionally treated counterparts, as showed in the MRI; this allowed them to resume full physical activity about 30% earlier than with conventional treatment. It is concluded that both methods are safe and effective for the treatment of muscle injuries. ACS may be prepared in advance in professional athletes so that it may be immediately available upon muscle trauma. Moreover, electrotransfer of genes may be used as a „silent preventive treatment” in susceptible muscle groups.

Abstract in German - Zusammenfassung:

Um neue Ansätze in der Behandlung von Muskelverletzungen zu untersuchen, wurden drei verschiedene Studien durchgeführt: (1) Mäusen wurde experimentell eine Kontusionsverletzung gesetzt, und aus ihrem Blut wurde ein autologes conditioniertes Serum (ACS) gewonnen, das mit Wachstumsfaktoren angereichert war. Wiederholte Dosen von ACS wurden likal in den verletzten Muskelbezirk injiziert; der zeitliche Verlauf der Regeneration wurde unter besonderem Augenmerk auf die Aktivierung der Satellitenzellen histologisch untersucht. (2) Athleten mit zweitgradiger Muskelzerrung erhielten eine Behandlung mit ACS (Kontrollgrupper mit konventioneller Behandlung), und ihr Heilungsverlauf wurde durch MRI beurteilt. (3) Unter Verwendung des gleichen Kontusionsmodells und histologischer Methodik wie in der ersten Studie wurde ein Elektrotransfer des FGF-2 Gens in die verletzten Muskelbezirke vorgenommen. Sowohl die Injektion von ACS wie auch Elektrogentransfer verbesserten das Ausmaß und den Zeitverlauf der Muskelheilung in vergleichbarem Umfang; dieser Befund gründet sich auf einer früheren und verstärkten Aktivierung der Satellitenzellen, was dem regenerierenden Muskel eine frühere Rückkehr zur strukturellen Integrität erlaubt.

Die mit ACS behandelten Athleten erholten sich schneller von der Muskelzerrung als die konventionell behandelten Sportler; objektiv konnte dies durch MRI verifiziert werden, und sie konnten die volle Trainingsbelastung ca. 30% früher wieder aufnehmen als die Kontrollgruppe. Es wird gefolgert, dass beide Methoden sicher und effektiv für die Behandlung von Muskelverletzungen sind. Es wird empfohlen, ACS von Leistungssportlern „auf Vorrat“ zu gewinnen, um es im Fall einer Muskelverletzung direkt verabreichen zu können. Darüber hinaus mag der Elektrotransfer von Genen eine präventive Behandlungsmethode in für Verletzung anfälligen Muskelgruppen darstellen.

Table of Contents:

	TABLE OF CONTENTS	7
	ABBREVIATIONS	9
	INTRODUCTION	10
	MUSCLE INJURIES	11
	OVERALL IMPORTANCE	11
	TYPES OF INJURY	11
	MUSCLE IMAGING: DIAGNOSIS AND FOLLOW-UP OF INJURIES	13
	REGENERATION PROCESS	15
	TIME FRAME OF REGENERATION	15
	SATELLITE CELLS IN REGENERATION	21
	GROWTH FACTORS IN REGENERATION	28
	CYTOKINES IN REGENERATION AND INFLAMMATION	37
	ANIMAL MODELS OF INJURY	38
	TREATMENTS	41
	CONVENTIONAL TREATMENTS	41
	ATTEMPTS WITH GROWTH FACTORS TO ACCELERATE REGENERATION	44
	AIMS OF THE STUDY	47
	AUTOLOGOUS CONDITIONED SERUM IN THE TREATMENT OF MUSCLE INJURIES	47
	ELECTROTRANSFER OF GENES INTO THE MUSCLE	49
	STRUCTURE OF THE THESIS	50
	MATERIALS AND METHODS	51
	TREATEMENT OF MUSCLE INJURIES WITH ACS: ANIMAL STUDY	52
	MICE	52
	CONDITIONED SERUM	52
	ELISA TESTS	53
	CONTUSION MODEL	54
	HISTOLOGICAL PROCEDURES: SATELLITE CELLS	54
	HISTOLOGICAL PROCEDURES: CENTRONUCLEATED MYOFIBERS	55
	FIBROSIS DETECTION	56
	STATISTICAL ANALYSIS	56
	TREATEMENT OF MUSCLE INJURIES WITH ACS: HUMAN PILOT STUDY	58
	AUTOLOGOUS CONDITIONED SERUM (ACS)	58
	ACS ELISA TESTS	59
	ACS SAFETY TESTS ON SERUM	59
	TREATED PATIENT GROUPS	59
	THERAPY REGIME AND EVALUATION OF RECOVERY	61
	STATISTICAL ANALYSIS	62
	ELECTROGENE TRANSFER INTO MOUSE MUSCLE	63
	CONSTRUCTION AND PURIFICATION OF PCMVBFGF (OR PVAXBFGF)	63
	PLASMID DNA	68
	MICE	69
	CONTUSION MODEL	69
	DNA INJECTION	69
	DNA ELECTROTRANSFER	70
	GFP EXPRESSION	71
	LUCIFERASE ACTIVITY MEASUREMENT	71
	QUANTIFICATION OF REGENERATION (SATELLITE CELLS)	72
	QUANTIFICATION OF REGENERATION (CENTRONUCLEATED MYOFIBERS)	73
	MEASUREMENT OF FGF-2 CONCENTRATION	74
	STATISTICAL ANALYSIS	74
	RESULTS	75
	TREATMENT OF MUSCLE INJURIES WITH ACS: ANIMAL STUDY	76
	ELISA RESULTS	76
	HISTOLOGY RESULTS	77
	QUANTITATIVE REGENERATION RESULTS	80
	TREATMENT OF MUSCLE INJURIES WITH ACS: HUMAN PILOT STUDY	87
	AUTOLOGOUS CONDITIONED SERUM GROUP	87
	CONTROL GROUP	89
	ELISA TESTS	91
	ELECTROGENE TRANSFER INTO MOUSE MUSCLE	92
	ELECTROTRANSFER LEVELS AFTER INJURY	92
	FGF-2 CONCENTRATION	93
	SATELLITE CELL ACTIVATION	95
	REGENERATING MYOFIBERS	97
	DISCUSSION AND PERSPECTIVES	100
	TREATMENT OF MUSCLE INJURIES WITH ACS: ANIMAL STUDY	101
	TREATMENT OF MUSCLE INJURIES WITH ACS: PILOT STUDY	104
	ELECTROGENE TRASFER INTO MOUSE MUSCLE	109
	GENERAL CONCLUSIONS	113
	SUMMARY	116
	BIBLIOGRAPHY	120