Charles S. Day

Growth factors improve muscle healing in vivo

Injury to muscles is very common. We have previously observed that basic fibroblast growth factor (b-FGF), insulin growth factor type 1 (IGF-1) and nerve growth factor (NGF) are potent stimulators of the proliferation and fusion of myoblasts in vitro. We therefore injected these growth factors into mice with lacerations of the gastrocnemius muscle. The muscle regeneration was evaluated at one week by histological staining and quantitative histology. Muscle healing was assessed histologically and the contractile properties were measured one month after injury. Our findings showed that b-FGF, IGF and to a less extent NGF enhanced muscle regeneration in vivo compared with control muscle. At one month, muscles treated with IGF-1 and b-FGF showed improved healing and significantly increased fast-twitch and tetanus strengths. Our results suggest that b-FGF and IGF-1 stimulated muscle healing and may have a considerable effect on the treatment of muscle injuries.

Peroneal nerve palsy following knee dislocation: pathoanatomy and implications for treatment

Abstract Peroneal nerve palsy following knee dislocation is a serious problem, and neurolysis at the time of knee reconstruction does not always result in return of peroneal nerve function. We describe peroneal nerve pathoanatomy in three patients in whom late exploration of the peroneal nerve was performed because of ongoing absence of ankle dorsiflexion. We identified frank nerve rupture in two patients and a lengthy neuroma in continuity in one which extended far proximal to the fibular head and well above the previous surgical incision used for peroneal nerve neurolysis at the time of knee reconstruction. In light of the current state of microneural surgery and the potential to reconstruct nerve defects, we discuss how our findings impact on treatment, and provide recommendations which may improve recovery of peroneal nerve function in future cases.

The efficiency of muscle-derived cell-mediated bone formation.

The development of new clinically applicable methods for the delivery of bone morphogenic protein (BMP) is an area of intensive research. Cell-mediated gene therapy approaches are being explored as a potential delivery vehicle. Primary muscle-derived cells isolated from an adult mouse were transduced with an adenoviral–BMP-2 construct. These cells were injected into the triceps surae of severe combined immune deficient (SCID) mice where they induced heterotopic bone formation. BMP-2 expression by these muscle-derived cell constructs was measured in vitro to estimate in vivo BMP-2 delivery. In vitro expression of BMP-2 by 3 × 105 muscle-derived cells was 87.89 ng/72 h. These results suggest that the efficiency of muscle cell-based gene delivery of BMP-2 exceeds the direct delivery of recombinant BMP-2 protein.

Gene transfer to muscle using herpes simplex virus-based vectors

The main goal of gene therapy for Duchenne muscular dystrophy (DMD) is to restore dystrophin into as many muscle cells as necessary to be therapeutic. Herpes simplex virus type 1 (HSV-1) represents a promising new viral vector capable of efficient transduction of myofibers in vivo. The viral genome is large and can accommodate multiple or large non-viral genes including the full-length dystrophin. Here we report on the use of a replication defective HSV-1 mutant vector (DZ) deleted for the essential immediate early (IE) gene ICP4 for studies of reporter gene transfer and expression following direct inoculation of mouse skeletal muscle. Our initial experiments showed that HSV-1 can efficiently infect and express a foreign reporter gene in myoblasts and myotubes in vitro. Furthermore, the intramuscular inoculation of HSV-1 resulted in transduction of a significant number of muscle fibers in newborn mice and some muscle fibers in adult animals. We have attempted to exploit these features to develop new HSV mutant vectors for dystrophin gene delivery to DMD muscle, however two impediments to using this virus for muscle gene delivery have to be overcome: namely viral cytotoxicity and the differential transducibility with HSV-1 throughout the development of muscle fibers. To solve the first problem, virus mutants deleted for the immediate early (IE) genes (ICP4, ICP22, ICP27 and UL41) were constructed and the multiple deleted virus was greatly reduced in cytotoxicity relative to our first generation HSV vector strains. Current work is aimed at incorporating full-length dystrophin under muscle specific promoter (muscle creatine kinase MCK) into these new viral vectors. To address the second problem we have analysed by immunohistochemistry the spreading of the HSV-1 in newborn versus adult muscles to determine whether mature basal lamina which surrounds the adult muscle fibers blocks the HSV-1 entry into the mature muscle fibers.

Biologic intervention in muscle healing and regeneration

Muscle injuries are a challenging problem in traumatology and the most frequently occurring injuries in sports medicine. Even though muscles retain their ability to regenerate after injury, the healing process of muscles after such injuries has been found to be slow and often leads to an incomplete muscle recovery. In an attempt to develop approaches to improve muscle healing after injury, the authors have developed reproducible injury models for muscle contusion, strain, and laceration. The authors show that muscle regeneration occurs after those injuries, but the development of scar tissue greatly limits the natural healing process. It is likely that an enhancement of muscle growth and regeneration can be used to improve muscle healing after injuries. The authors have then identified growth factors that enhance myoblast proliferation and differentiation in vitro and muscle regeneration in the injured muscles, which improves muscle healing after injuries. Furthermore, different gene transfer systems, including cell and gene therapy, have been found successful in delivering genes into injured muscles and may open new opportunities to deliver growth factors and improve muscle healing after lacerations, contusions, and strains.

Muscle-Based Tissue Engineering for Orthopaedic Applications

The advent of gene therapy and tissue engineering has facilitated novel approaches to the treatment of orthopaedic disorders. The delivery of growth factors, cells, and therapeutic genes promises therapeutic possibilities not contemplated by previous generations of orthopaedic surgeons. Significant scientific contributions have been made in the last 3 decades toward the understanding of skeletal muscle biology and its potential therapeutic applications. However, despite tremendous progress, many questions remain unanswered. This chapter reviews the current status of muscle-based tissue engineering for musculoskeletal disorders and discusses the focus of ongoing research.

The Development of Approaches Based on Gene Therapy to Improve Muscle Healing Following Injury

Muscle injuries are common, with an incidence varying from 10% to 55% of all injuries sustained in sports (Lehto and Jarvinen 1991). Muscle injuries are divided into 2 types: a shearing injury, in which both the myofibers and the connective tissue framework are torn, or an in situ injury, in which only the myofibers are damaged and the basal lamina and connective tissue sheaths do not undergo significant harm. Shearing injuries, the most frequent muscle injuries related to sports, may be lacerations, contusions, or strains, depending on the mechanism of injury (Lehto and Jarvinen 1991). Contusion is sustained through a significant compressive force to the muscle, such as a direct blow, a common occurrence in contact sports. A strain occurs when a forceful eccentric contraction is applied to an overstretched muscle, especially in jumping or sprinting (Garrett, Jr. 1990; Lehto and Jarvinen 1991). Injury is common near the musculotendinous junction (MTJ) of a superficial muscle that crosses 2 joints, such as the rectus femoris, semitendinosus, and gastrocnemius muscles. Though rather rare in sports, muscle laceration is a dramatic injury that consistently incapacitates athletes for long periods of time and often jeopardizes their professional careers.