We evaluated the efficacy of treating eccentric muscle damage with IGF-1 PEGylated to a fibrin biomatrix. With one injection, delivered one hour after the induction of eccentric muscle damage we saw an attenuation of force loss early in recovery, maintenance of muscle weight, and progression to the repair/regeneration of the damaged fibers at a greater speed and magnitude in the first week of recovery. As opposed to introducing an unbound bolus of IGF-1, we believe the ability of the PEGylated-fibrin to stabilize and sustain delivery of the molecule results in significantly better recovery. Coupling IGF-1, which has multiple beneficial effects in tissue repair, with this system of delivery provides a simple and easy to administer treatment for eccentric muscle damage. With this form of damage being the most prevalent of all skeletal muscle damage types, since it is underlies all muscle strain, a simple and effective treatment is important for increasing functional recovery after injury. / text

The toll skeletal muscle injury, resulting in significant muscle mass loss, has on the patient reaches far more than physical and emotional, as the tolls are financial as well. Approximately more than 3 billion dollars is spent on the initial medical costs and on subsequent disability benefits, following a volumetric muscle loss. Skeletal muscle has a robust capacity for self-repair; this propensity for repair is hindered when skeletal muscle loss is larger than 20% of the total mass of the muscle. Previous work in our lab, has shown functional and morphological improvements following the cellular therapy, with mesenchymal stem cells (MSC), as well as with nerve relocation to the extracellular matrix (ECM). To further observe the regenerative properties of the above treatments, a defect weighing approximately 307 ± 3.7 mg wet weight and measuring approximately 1x 1cm² was removed from the lateral gastrocnemius (LGAS) of male Sprague Dawley rats. Additionally, the medial branch of the plantar nerve was then relocated and implanted to the middle of the ECM. Seven days post injury bone-marrow derived mesenchymal stem cells were injected directly into the implant using a PEGylated Fibrin hydrogel (PEG). Following 56 days of recovery, partial functional restoration was observed in the LGAS ECM seeded with MSC and implanted with the plantar nerve. The LGAS produced 86.3 ± 5.8% of the contralateral LGAS, a value that was significantly higher than ECM implantation alone (p <.05). The implanted ECM seeded with MSC and implanted with the plantar nerve showed significant increases in blood vessel density and myofiber content (p <.05). The data suggest that a volumetric injury can be repaired by neurotization of an implanted muscle-derived ECM seeded with MSCs. / text

This study investigated the effect of bone marrow derived mesenchymal stem cells (MSCs) in a PEGylated fibrin gel (PEG) seeded into a decellularized extracellular matrix (ECM) on recovery of skeletal muscle following a volumetric muscle loss (VML) injury. Six to nine month old male Sprague-Dawley rats were used in this study. Approximately one-third of the skeletal muscle mass of the lateral gastrocnemius (LGAS) was removed from the LGAS, which was immediately replaced with an acellular ECM of the same dimensions. Seven days after injury, animals were injected with one of four solutions: saline (SAL), MSCs (MSC), PEGylated fibrin hydrogel (PEG), or MSCs in PEG (PEG+MSC). Maximal isometric tetanic tension (Po) of the LGAS was assessed fifty-six days after VML injury, followed by histological evaluation. VML injury resulted in a functional impairment of the LGAS capable of producing 76.1± 4.9% of the force generated in the non-injured contralateral LGAS. Tetanic tension of the PEG+MSC treated group was significantly higher compared to all other treatment groups (p < 0.05), although specific tension (N/cm2) in the PEG+MSC group (79.7±4.0%) was only significantly higher compared to SAL (58.2±3.0) and PEG (64.0±2.1%) treated groups (p < 0.05). However, LGAS mass was significantly higher in the PEG+MSC group compared to all other groups (p < 0.05). These findings suggest the combination of the PEG+MSC did not lead to a significant increase in muscle function compared to MSC treatment alone, and demonstrates the importance of MSCs in skeletal muscle regeneration in VML injury models. However, as evident by the significant increase in LGAS mass, PEG+MSC treatment may lead to histological differences not evaluated in this study. Gross morphology of the repaired gastrocnemius was indistinguishable from the contralateral control. / text

Ischemia/reperfusion (I/R) injury causes extensive damage to skeletal muscle, often resulting in prolonged functional deficits. This current study determines the efficacy of controlled release of SDF-1α and IGF-1 by conjugation to biodegradable, polyethylene glycol, (PEG)ylated fibrin gel matrix in skeletal muscle repair of an I/R injury. Male Sprague-Dawley rats underwent a 2-hour tourniquet induced I/R injury on their hind limbs. Twenty-four hours post injury the following treatments were administered: PEGylated fibrin gel (PEG-Fib), SDF-1 conjugated PEGylated fibrin gel (PEG-Fib/SDF-1), or dual protein IGF-1 and SDF-1 conjugated PEGylated fibrin gel (PEG-Fibrin/SDF-1/IGF-1. Following 14 days after injury, functional and histological evaluations were performed. There was no significant difference in maximum tetanic force production recovery between PEG-Fib and PEG-Fib/SDF-1 groups. However, PEG-Fib/SDF-1/IGF-1 group resulted in significant improvement of force production relative to the other treatment groups. The same results were found for specific tension. Histological analysis revealed a greater distribution of small myofibers in the PEG-Fib/SDF-1 group than the PEG-Fib group, while the PEG-Fib/SDF-1/IGF-1 group had the smallest distribution of small fibers and similar to controls (uninjured). There were also a greater number of centrally located nuclei in the PEG-Fib/SDF-1 group than the PEG-Fib group, while the PEG-Fib/SDF-1/IGF-1 group had similar values to controls. Although these results confirm the protective role of exogenous IGF-1, SDF-1 did not have an effect on skeletal muscle repair. / text

Tourniquet mediated Ischemia Reperfusion (I/R) injury causes damage to skeletal muscle, often resulting in prolonged functional impairment. The current study utilizes immunohistochemistry (IHC) to determine whether the controlled release of the anabolic factor, insulin-like growth factor-I (IGF-I), from the biodegradable PEGylated fibrin gel matrix can facilitate the recovery of skeletal muscle from I/R. Treatment groups following a 2-hour tourniquet applied to the limb of 6-9 month rats, included intramuscular injections of saline, PEGylated fibrin gel (PEG-Fib) only and IGF-I conjugated to PEGylated fibrin gel (PEG-Fib-IGF). Expression of the myogenic regulatory factors MyoD and myogenin detected via IHC in the PEG-Fib-IGF group was significantly lower compared to the saline group, showing a 1.4±0.8% nuclear co-localization for MyoD and a 2.0±0.8% nuclear co-localization for myogenin at 14 days of recovery. The saline group showed higher values, 31.4±4.4% and 44.1±7.3% for MyoD and myogenin nuclear co-localization respectively. A significantly greater percentage, 88.8±3.7% of Desmin positive myofibers was seen at 14 days of recovery, while a lower percentage of fibers expressing neonatal myosin, 7.7±2.7% was seen in the PEG-Fib-IGF group compared to the saline treatment group. These results indicate that IGF-I delivered intramuscularly via PEGylated fibrin gel, functions therapeutically in skeletal muscle recovery, from I/R mediated damage. In a separate injury model that deals with volumetric muscle loss, IHC analyses were performed to test the efficacy of a novel tissue engineering strategy utilizing extracellular matrix (ECM) as a scaffold. In this model, also called the defect model, a 1.0 X 1.0 cm piece of the lateral gastrocnemius was removed and replaced with a muscle-derived ECM. The constructs were then seeded with bone marrow derived cells (BMSCs), adipose derived stem cells (ADSCs) or the peroneal nerve was relocated to the area of the ECM implant. 42 days post recovery IHC analysis was performed on the ECM implants. The quantification of desmin-positive regenerating myofibers bearing centrally located nuclei, showed significantly greater values in the top, middle and bottom region of the ECM implants that received peroneal nerve relocation, when compared to the experimental group that received the ECM implant alone. Blood vessel density increases were seen within the middle region of the ECM implant groups that received BMSC+Nerve treatment and the bottom region of the ECM implant groups that received ADSC+Nerve treatment. Thus, these results corroborate the therapeutic effect of peroneal nerve relocation, which stimulated an increase in myofiber regeneration and vascular maintenance within the construct. / text