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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session D

Poster V13 - A Combined Tissue-Engineered, Immunomodulatory, and Rehabilitative Therapy for Volumetric Muscle Loss

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Harper McClain (she/her/hers)

Undergraduate Researcher
University of Oregon, Phil and Penny Knight Campus for Accelerating Scientific Impact, Wu Tsai Human Performance Alliance
Eugene, Oregon, United States

Primary Investigator(s)

NW

Nick Willett, PhD

Associate Professor
University of Oregon - Knight Campus, United States

Co-Author(s)

FP

Frank Pittmen, MS (he/him/his)

BioE Ph.D. Student
University of Oregon, United States
KL

Kait Link, DVM

Research Associate, Wu Tsai Human Performance Alliance
University of Oregon, United States
Christine Tran, BA

Post-Baccalaureate
University of Oregon
Wichita, Kansas, United States

Introduction: VML is a significant clinical burden that results in long-term functional deficits such as chronic pain and loss of muscle strength and mobility, as well as fibrosis, fatty infiltration, and inflammation. Standard care for VML includes autologous muscle flap transfer followed by exercise rehabilitation. This procedure can result in complications such as immune rejection, insufficient vascularization, fibrosis, contractures and poor innervation. Furthermore, rehabilitation-mediated functional improvements after VML are limited. A dysregulated immune response after VML injury has been shown to impair myogenesis and exacerbate fibrosis, ultimately leading to muscle wasting. These effects are partially mediated through Transforming Growth Factor β1 (TGF-β1) overexpression by a dysregulated macrophage population. TGF-β1 has been linked to a number of muscle pathologies that result in fibrotic accumulation such as ALS and muscular dystrophy. We have previously investigated the specialized pro-resolving mediator (SPM) Resolvin D1 (RvD1) for its ability to attenuate pro-fibrotic signaling and enhance muscle regeneration by resolving chronic inflammation in the VML injury niche. However, there is little investigation into the co-treatment of VML with pro-resolving mediators with the clinically used paradigm of tissue transfer and exercise rehabilitation. Our overall objective is to improve functional muscle regeneration in VML-injured muscle using a novel therapy that synergizes tissue-engineered muscle constructs with immunomodulatory SPMs and exercise. Our overall hypothesis is that AT-RVD1 will foster a pro-regenerative microenvironment in the VML injury to enhance engraftment of a tissue-engineered muscle construct for improved functional muscle regeneration. Additionally, we hypothesize exercise rehabilitation will further enhance functional muscle recovery.

Materials and

Methods: Male C57BL6/J mice (8-12 week) received a subcritical, healing muscle defect via a 2 mm biopsy punch or a critical, non-healing defect via 3 mm size biopsy punch to obtain a VML quadriceps injury. All work was conducted under local approved IACUC protocols.

Animals were pre-trained on a voluntary running wheel prior to surgery, and then the running wheel was reintroduced to exercising groups 7 days post-VML until the study endpoint at week 4.

Quadriceps muscle strength was assessed ex vivo by isometric torque about the knee using electrical stimulation on the femoral nerve. Dynamic weight bearing (DWB) was used to quantify spontaneous pain behavior by measuring time duration spent on each limb.

Murine C2C12 myoblast differentiation was induced by treatment with 5% heat-inactivated horse serum and applied TGF-β1 dosages with the co-delivery of AT-RvD1. Media was replenished every 48 hours until staining for MHC expressing myotubes.

Primary myoblasts and C2C12s were stained with a rabbit anti-myosin heavy chain primary antibody overnight, followed by a Goat anti-rabbit IgG H&L (alexa flour 594) secondary antibody and DAPI. Cells were imaged on a Leica Thunder fluorescence microscope at 5X to analyze myotube formation and nuclear fusion index in ImageJ. Statistics were performed in GraphPad Prism 9. To analyze C2C12 TGF-β1dosage response a one-way ANOVA of multiple comparisons with a Tukey post-hoc pairwise comparisons (95%) was used and a two-tailed Unpaired T-test for primary myoblasts was used.

Results, Conclusions, and Discussions: Myoblasts were cultured on collagen sponges where the cells proliferated, differentiated and fused to form MHC expressing myotubes. Cell seeded collagen scaffolds were implanted into a murine of quadriceps VML defect. Post-injury, there was an increase in pain observed from baseline to week 3 in treatment groups receiving cell-laden collagen scaffolds measured by DWB. In isometric torque analysis at week 4, decreased muscle functionality was observed when comparing the average isometric torque of the injured limb receiving cell-laden collagen scaffolds compared to the average isometric torque of the contralateral. Further, exercise rehabilitation in the form of voluntary wheel running did not improve muscle mass or strength outcomes as measured by muscle wet weight at takedown and isometric torque about the knee at week 4. In separate cell culture studies, the effects of TGF-β1 (an anti-myogenic and pro-fibrotic condition) and the pro-resolving lipid mediator, AT-RvD1, were investigated in both primary and C2C12 cell line myoblasts. Immunofluorescent imaging from the cell culture studies showed that AT-RvD1 increased myotube formation in C2C12 cells and in primary myoblasts when cultured in anti-myogenic and pro-fibrotic conditions (Fig. 1A **p=0.0061, 1A *p=0.0264, 1B **p=0.0088). Together, these results indicate an opportunity for future work to investigate the co-treatment of VML injuries with both myoblast-laden collagen scaffolds and AT-RvD1.

Acknowledgements (Optional):