Introduction:: Volumetric muscle loss (VML) is a condition in which approximately 20% or more of a skeletal muscle’s mass has been destroyed. VML is characterized by lack of muscle regeneration and loss of functionality1. The current treatment for VML is an autologous tissue transfer, which often results in graft failure2. Tissue engineering seeks to develop implantable scaffolds which can regenerate an injured muscle and restore functionality. Native muscle tissue contains aligned myofibers; thus, cells infiltrating a scaffold should orient in a single direction to form aligned myofibers, leading to restoration of functionality. To our knowledge, the effect of pore size on myofiber formation has not been directly studied. We seek to develop an aligned collagen scaffold with optimal pore size for myoblast behavior to generate an off-the-shelf, implantable scaffold to use as a treatment for VML.
Materials and Methods:: Type I rat tail collagen (5 mg/mL) was polymerized into hydrogels within cylindrical PTFE molds. Directional freezing was achieved by placing these hydrogels on a metal block pre-chilled to -20, -40, -60, -80, or -196°C and incubating until frozen. Frozen gels were lyophilized to form anisotropic sponges. The sponges were sectioned, stained, and analyzed using light microscopy and ImageJ to quantify pore diameters. To quantify myoblast differentiation, 1 million cells/cm2 were seeded onto each side of the collagen sponges, cultured for 10 days, then fixed in paraformaldehyde and cryosectioned. Sections were stained with MF-20, phalloidin, and DAPI. Total cell numbers and the pore and cell angles were quantified using ImageJ. Percent infiltration was calculated based on cell count. Statistical analyses were conducted with one-way ANOVA with Tukey’s post-hoc analysis (p< 0.05).
Results, Conclusions, and Discussions:: Results: Representative histology images from scaffolds frozen at -20 (Fig 1A), -40 (Fig 1B), (Fig 1D) -60, (Fig 1E) -80, and (Fig 1F) -196°C reveal evenly distributed pores throughout the cross-section of the scaffolds. Pore diameter measurements reveal that pore diameter decreases with decreasing freezing temperature (Fig 1C). Quantification of cell infiltration (Fig 1I) reveals a qualitatively higher percentage of cell infiltration in -20°C sponges as compared to other freezing groups. While -80°C sponges also had higher cell infiltration, the average pore angle and cell angle within these scaffolds differed significantly. Cell angle (Fig 1H) and pore angle (Fig 1J) measurements reveal that most cells oriented between 75-85°, while pores oriented between 85-95°. These data also show that -20°C scaffolds have the most pore orientation near 90°, which indicates axial alignment. Additionally, most cells in the -20°C scaffolds oriented themselves in the range of 85-95°, which indicates cell alignment around the same angle of pore alignment. Myoblasts infiltrated and differentiated into myofibers (Fig 1G) within these pores, suggesting that these materials would facilitate muscle regeneration in situ.
Conclusions: These data confirm that scaffold freezing temperature changed the pore size within the scaffold. We have established a method of creating scaffolds with tunable pore size for specific tissue engineering applications. We found that a pore size of 88.9 ± 17.6 µm, made by freezing the collagen at -20°C, enhanced myoblast infiltration. Similar pore and cell angle distributions within scaffolds frozen at -20°C indicate that the cells were oriented in the same direction as the pores, as shown in our histology. Additionally, we see that most pores in the -20°C scaffolds were oriented toward a 90° angle. These findings identify processing parameters that are ideal for skeletal muscle regeneration, as myoblasts must be aligned in order to transmit uniaxial force to restore functionality. Future in vivo studies will be conducted to determine the regenerative capabilities of these scaffolds in a muscle injury.
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References (Optional): : 1. Grasman, J.M. et al. Acta Biomater. 2015;25:2-15. 2. Mulbauer, G.D. et al. Discoveries (Craiova). 2019;7(1):e90.
