(A-32) Fibrin Scaffolds with Anisotropic Pores Display Tunable Mechanics that Modulate Myotube Differentiation for Skeletal Muscle Regeneration

Introduction:: Introduction: Volumetric muscle loss (VML) is a condition 65.8 million Americans suffer from annually [1], including a disproportionate number of wounded warriors injured in combat [2]. VML is caused when significant skeletal muscle loss damages the biophysical and biochemical signaling cues that facilitate functional muscle regeneration. Tissue engineering strategies that use biomimetic scaffolds offer a promising solution for treating VML by providing anisotropically-aligned topographic features to guide myotube alignment which is important to produce uniaxial contractile forces. Recently, our lab developed a directional lyophilization technique to create fibrin scaffolds, where the anisotropic porosity can be tuned by altering the fabrication parameters. We showed that anisotropic scaffolds with decreased freezing temperatures result in maximized myoblast orientation. Here, we aim to characterize myotube differentiation efficiency on our tunable scaffolds. Native muscle tissue ranges from 5 to 15 kPa, and substrates adjusted to that stiffness have been shown to increase the differentiation and regenerative capacity of myoblasts [3]. The objective of this study is to determine how our fabrication parameters impact scaffold mechanics, and consequently myotube formation. As such, we hypothesize that higher scaffold concentrations will increase stiffness and that scaffold mechanics similar to normal muscle tissue will improve myoblast differentiation.

Materials and Methods:: Materials and Methods: Scaffolds were fabricated by mixing fibrinogen, thrombin, and CaCl₂ to produce 10 or 20 mg/mL fibrin solutions. Anisotropy was introduced by adding the fibrin solutions to custom wells with conductive aluminum plates and insulated PDMS walls to produce a vertical freezing gradient when placed on pre-frozen surfaces. These fibrin solutions were polymerized for 30 minutes, rinsed, and placed on frozen surfaces at pre-set temperatures (-40, -80, or -195°C) for 15 minutes. Frozen scaffolds were lyophilized at -15°C for 2 days, then sectioned along the longitudinal or transverse planes for testing (Figure 1A). Mechanical characterization was performed using an Optics11Life Chiaro Nanoindenter with a 269μm spherical tip. Effective Young’s moduli were calculated using the Hertz model in the Data Viewer software. Myotube formation was evaluated by seeding C2C12 myoblasts onto anisotropic scaffolds (2 million cells/mL). Cells were switched to differentiation medium after 2 days and cultured for 7 additional days. Samples were fixed and stained with myosin heavy chain (MHC) and Hoechst, then imaged with fluorescence microscopy to visualize the myotube bodies and nuclei. Cell Profiler was used to quantify nuclear fusion index and myotube length.


Results, Conclusions, and Discussions:: Results and Discussion: 20 mg/mL scaffolds had significantly increased moduli (2.19 kPa to 4.77 kPa) compared to 10 mg/mL scaffolds (0.21 kPa to 0.72 kPa) in all temperature groups (Figure 1B). These values indicate that the 20 mg/mL scaffolds are closer in stiffness to that of the native tissue. Freezing temperature did not appear to impact scaffold stiffness. While the transverse samples displayed a general trend of larger moduli compared to longitudinal samples for each condition, there was no significant difference. Myotube formation, measured by MHC staining, was observed in all conditions, however, 20 mg/mL scaffolds displayed higher levels of expression. However, the general expression levels on all scaffolds were low, so cells will be differentiated for longer in the future. Cells on non-directional controls (TCP) showed no preferred orientation, while cells on anisotropic scaffolds showed an alignment preference to the scaffold orientation (Figure 2). Quantitative analysis for myotube morphology is ongoing.

Conclusions: Directionally frozen scaffolds provide topographical cues that direct myotube formation and alignment. The higher concentration scaffolds result in matrix mechanics similar to native tissue, which appears to increase myotube formation. Future work will focus on quantification of myotube contraction, as well as the integration of vascular networks and biochemical signaling to our scaffolds.


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References (Optional): : References: [1] M.E. Carnes and G.D. Pins, Bioengineering, 2020, 7(85).; [2] S. Testa et al., Biomedicines, 2021, 9(564).; [3] A.J. Engler et al., Journal of Cell Biology, 2004, 166(6).