Z19 - Extrusion-Based Printing of Myoblast-Loaded Fibrin Microthreads to Induce Myogenesis

Introduction: : Approximately 65.8 million Americans suffer from musculoskeletal injuries every year that can result in volumetric muscle loss (VML) and functional muscle deficits [1]. The current standard of care for VML injuries consists of free muscle flap transfers (FFTs), which transfer an innervated vascularized donor muscle to the wound site, to facilitate tissue regeneration [2]. This procedure is limited by tissue supply, donor site morbidity and scar tissue formation [3]. Further, this treatment exhibits limited tissue innervation and poor functional muscle regeneration. Thus, there is an unmet need to produce an implantable skeletal muscle construct that recapitulates the native tissue niches [3],[4]. Our laboratory developed fibrin microthreads that confer native muscle architectural and regenerative cues that promote cell-mediated myogenesis [4]. Additionally, cells such as myoblasts are required to replenish the necessary cell population for skeletal muscle tissue regeneration [5],[6]. As such, we hypothesize that increasing the myoblast densities in extruded fibrin microthreads (myothreads) will enhance myogenic properties including increased cell alignment, myotube formation, and reproducible mechanical properties of the skeletal muscle constructs.

Materials and

Methods: : C2C12 mouse myoblasts were cultured in proliferation medium (CPM; 1:1 DMEM/F-12 medium, 10% Fetal Bovine Serum; FBS, 1% P/S: Pen-Strep) until the cells were up to 70% confluent. Varying densities of C2C12s (1-4 x 10^6 cells/mL) were incubated at 37ᵒC for 2 hours and mixed with 10 mg/mL fibrinogen in 20 mM HEPES buffer saline (HBS) buffer. Myothreads were fabricated by co-extruding C2C12s loaded fibrinogen and 6 U/mL thrombin solutions containing 6 mM CaCl2 into a sterile filtered 50% DMEM and 25% DPBS extrusion buffer. After 15 minutes of polymerization, myothreads were incubated in CPM with 20 µg/mL aprotinin for 3 days; then differentiated in differentiation medium (CDM; 1:1 DMEM/F-12, 2% Horse Serum; HS, 1% Insulin Transferrin Selenium; ITS, 1% P/S, 20 µg/mL aprotinin) for 6 days. Medium was changed every 2 days. C2C12-loaded fibrin gels were used as controls. Myothreads were stained with Hoescht, Phalloidin, and myosin heavy chain (MHC) to analyze nuclear alignment and myotube formation. Uniaxial tensile testing was used to characterize mechanical properties of the myothreads.

Results, Conclusions, and Discussions:: Analyses show that there is no statistical difference in nuclear alignment among myothreads with varying myoblast densities, but myothreads exhibited significantly higher alignment than cell-seeded fibrin gels (Figure 1A). Increasing myoblasts densities in myothreads enhanced myotube formation. Myothreads with 4x10^6 cells/mL had the greater MHC+ nuclei percentage and myotube dimensions (length, diameter, and area), than myothreads with 1x10^6 cells/mL. Fibrin gels had lower MHC+ nuclei percentage but myotube dimensions comparable to 2x10^6 and 4x10^6 cells/mL myothreads. Finally, myothreads with 1x10^6 cells/mL did not have statistically significant differences from fibrin gels in MHC+ nuclei and myotube diameter but had significantly decreased myotube length and area (Figure 1B-F). Initially, myothreads did not exhibit any statistical differences in elastic moduli, UTS, and SAF as a function of myoblast densities (Figure 1H-J). However, culturing myothreads for 9 days significantly decreased elastic moduli from ~400 kPa to ~170-190 kPa, and culturing myothreads with 4x10^6 cells/mL significant decreased in UTS relative to acellular fibrin microthreads (Figure 1H, I). These studies suggest that cell-loaded myothreads may induce skeletal muscle development through nuclear alignment, and myotube formation, as well as mechanical properties similar to values of fibrin-based skeletal muscle constructs [7].

Our findings illustrate that co-extrusion of myoblast-loaded fibrin micothreads can confer greater nuclear alignment in myothreads relative to fibrin gels and increasing myoblasts densities in myothread extrusion can induce myogenic properties; specifically myotube formation as well as tensile properties comparable to previously published skeletal muscle constructs [7]. We anticipate that these novel myothreads may serve as an in vitro model to evaluate skeletal muscle regeneration through cocultures with neuronal cells and therapeutic agents.

Acknowledgements and/or References (Optional):: Acknowledgements: The authors acknowledge support from the WPI TRIAD Seed Grant program.

References: [1] Bone and Joint Initiative USA. Musculoskeletal Injuries. United States. 2016. [2] Garg et al. J. Orthop. Res. 2015; 33, 40. [3] Gilbert‐Honick, J., and Grayson, W.. Adv Health Mat. 2020; 9,1. [4] Carnes et al. Bioeng. 2020; 7,85. [5] Langridge et al. J Mat Sci Mater Med. 2021; 32, 15. [6] Gahlawat et al. Ann Biomed Eng. 2024; 52, 2325-2347. [7] Fan et al. Biofab. 2022; 14, 015009.