Introduction:: Volumetric muscle loss (VML) injuries are large skeletal muscle injuries characterized by a permanent loss of function and at least 20% of the muscle volume [1]. For injuries this large, the innate repair mechanism of skeletal muscle cannot fully regenerate the lost muscle, and thus, these injuries significantly impact patient quality of life. Therefore, implantable scaffolds can be considered a viable alternative to facilitate functional regeneration of muscle tissue after VML [1]. Scaffold stiffness has been shown to impact myoblast proliferation and differentiation into functional muscle tissue: softer scaffolds promote myoblast growth and differentiation, but if a scaffold is too soft or weak, the scaffold may lose its structural integrity and fail [2]. Therefore, this project seeks to optimize the mechanical properties of fibrin sponges through crosslinking to delay degradation and ultimately enhance regeneration. Additionally, we seek to observe how crosslinking affects viability of myoblasts seeded onto these scaffolds to determine whether these fibrin sponges facilitate myofiber formation.
Materials and Methods:: Fibrin (10 mg/mL) was polymerized within PTFE molds to form hydrogels, which were then placed in a custom apparatus for anisotropic freezing at -20°C, followed by lyophilization to form sponges. To determine the impact of crosslinking on mechanical properties, sponges were crosslinked with either 12.9 mM EDC and 10.4 mM NHS (“Low EDC”), 200 mM EDC and NHS (“High EDC”), or 1 mM genipin (“Genipin”). Sponges were subjected to tensile testing at a strain rate of 5 mm/min and relevant mechanical properties were compared to uncrosslinked control sponges. To inhibit fibrinolysis, sponges were seeded with C2C12 myoblasts and treated with aprotinin (a fibrinolysis inhibitor) at concentrations of either 5, 10, 30, 50, or 100 µg/mL in myoblast growth medium. An optimal concentration of aprotinin (30 µg/mL) was determined and used to preserve sponge integrity for cell metabolism studies. Myoblasts were seeded onto crosslinked sponges at a concentration of 1 million cells/cm2 per side and cultured with aprotinin supplemented growth medium. CCK8 cell metabolism assays were conducted on days 1, 3, and 10 to determine the effect of crosslinking on cell metabolism.
Results, Conclusions, and Discussions:: Results of tensile testing show that Genipin and Low EDC sponges had significantly higher Young’s moduli than uncrosslinked controls (Fig 1A), as well as qualitatively higher load at failure (Fig 1D) and UTS values (Fig 1B). High EDC sponges also had significantly lower strain at failure values than uncrosslinked controls (Fig 1C). Results of degradation testing revealed that 30, 50, and 100 µg/mL aprotinin treatment all significantly slowed degradation as compared to 0, 5 and 10 µg/mL aprotinin. An aprotinin concentration of 30 µg/mL was determined to be optimal, as it was the lowest concentration that still delayed degradation. There was a qualitative increase in cell metabolism from Day 1 to Day 3 in all groups except Genipin, suggesting that genipin crosslinking may inhibit C2C12 metabolism (Fig 1E). Additionally, all groups decreased in cell metabolism from Day 3 to Day 10, potentially suggesting that cells may have detached from the scaffold, or that crosslinking may have negative effects on cell viability after long incubation times (Fig 1E). Our findings demonstrate our ability to tune mechanical properties of fibrin sponges via crosslinking. Aprotinin at 30 µg/mL was found to effectively inhibit fibrinolysis of fibrin sponges and was used in further cell studies. CCK8 assays demonstrated that crosslinking may affect the metabolic activity of C2C12 myoblasts seeded on fibrin sponges. These data suggest that Low EDC conditions may facilitate enhanced cell proliferation than genipin crosslinking and should be further researched for skeletal muscle regeneration. Further studies will seek to determine whether these crosslinking conditions induce apoptosis, or whether cells migrate out of the scaffolds. This research helps to design an optimized fibrin scaffold for skeletal muscle regeneration.
Acknowledgements (Optional): : This work was supported by the NIH (KL2-TR003018 & R21-AR079708) and by startup funds provided by NJIT.
References (Optional): : 1. Grasman, J et al. in Acta Biomater. (2015). 2. Romanazzo, S et al. in Sci Technol Adv Mater. (2012).
