(L-457) Effect of ECM Proteins on Force Production of Hydrogels Seeded with Myocytes

Introduction:: Skeletal muscle injuries account for 77% of healthcare visits in the United States and are the leading cause of disabilities amongst active soldiers [1, 2]. While minor injuries regenerate, large injuries, termed volumetric muscle loss (VML), will not if more than 20% of the muscle is lost, resulting in permanent loss of function [3]. Treatments such as physical therapy and autologous muscle transplant aid in recovery but are limited by availability, tissue failure, and are incapable of restoring full functionality [3]. Tissue-engineered skeletal muscle may provide a treatment for VML that is not dependent on available muscle tissue and, by tailoring additives used within these constructs, may improve functional recovery. While Matrigel is the standard for fabricating muscle in vitro, there have been no systematic studies evaluating the effect of extracellular matrix (ECM) proteins on muscle maturity and force production, nor studies comparing the relationship between ECM and myofiber alignment. We hypothesize that the addition of exogenous ECM proteins found in the basal lamina of skeletal muscle in collagen gels seeded with primary rat myoblasts will improve myofiber differentiation and tissue compaction force and, when cultured in a static system, will increase myofiber alignment as a result of this higher force production.


Materials and Methods:: To determine the effects of ECM proteins on tissue force production, we seeded collagen hydrogels with myoblasts isolated from adult rat skeletal muscle and used a novel bioresin device to measure hydrogel compaction. Based on preliminary results utilizing C2C12 myoblasts, gels were supplemented with fibronectin and cultured in differentiation medium for nine days. Hydrogels supplemented with Matrigel or no exogenous ECM proteins served as our positive and negative controls, respectively. A bio-resin device consisting of an immobile resin peg and a mobile polydimethylsiloxane (PDMS) peg was used to measure changes in force using cantilever mechanics. After nine days, gels were fixed, immunostained against myosin, and counterstained with phalloidin and DAPI. Myofiber fusion index was calculated, and alignment was determined via ImageJ. Since deflection of the mobile peg reduces strain on the hydrogel, the experiment will be repeated with a static peg model as to observe the effect of ECM protein mediated compaction on strain-induced myofiber alignment.

Results, Conclusions, and Discussions:: Fibronectin loaded hydrogels seeded with primary myoblasts exhibited significantly higher compaction forces starting on day five and remained stable for the remainder of the study (Fig. 1 A). Forces were their highest on day five with a measured force of 88.3±15.6μN. Compaction force for control gels was highest on day 6 at 45.8±17.1μN, and were consistently lower than fibronectin hydrogels. Mobile peg displacement (Fig 1B) followed a similar trend, with fibronectin-treated hydrogels exhibiting significantly more displacement of the mobile peg starting at day five. In separate studies where control hydrogels were seeded with C2C12s (Fig 1C), the use of a static system significantly enhanced myofiber alignment, with nearly 45% of all measured fibers falling within 0-10 degrees of the centerline of the tissue (Fig. 1F). Hydrogels cultured with a mobile peg (Fig. 1E) or with no device at all exhibited only 16% and 11% of fibers between 0-10 degrees of the centerline respectively (Fig. 1D). These data demonstrate that ECM proteins impact not only myofiber maturation and force production in 3D collagen hydrogels, but that the strain produced by this force plays role in myofiber organization as well. Moving forward, we aim to repeat these studies with a mechanical system that actively stretches and relaxes the tissues in order to explore the effect that repetitive mechanical strain has on myofiber alignment and maturation


Acknowledgements (Optional): : This study has been funded in part by NJIT startup funds and the NIH (KL2-TR003018)

References (Optional): : [1] “By the Numbers; musculoskeletal injuries” US Bone and Joint Initiative (2012).
[2] B.T. Corona, “Volumetric muscle loss leads to permanent disability following extremity trauma” JRRD 52(7) (2015) 785-792.
[3] Hurtgen. BJ, S., “Severe muscle trauma triggers heightened and prolonged local musculoskeletal inflammation and impairs adjacent tibia fracture healing” Musculoskelet Neuronal Interact 16(2) (2016) 122-134