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Tissue Engineering

Tissue Engineering - Poster Session A

Poster L2 - Composite Tissue Engineered Approach Based on Microfiber Scaffolds to Resist Hydrogel Contraction

Thursday, October 24, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Volumetric muscle loss (VML) injuries, resulting from blast trauma, car accidents, or tumor excision, often exceed innate muscle regeneration capabilities and result in diminished functional outcomes. Tissue engineering (TE) brings together cellular delivery, therapeutic delivery, and alignment cues via scaffolds or matrices and has the potential to treat conditions such as VML. One challenge facing TE is the contraction of soft matrices used to encapsulate cells. Using Type-1 collagen combined with aligned melt electrowriting (MEW) microfibers provides a composite tissue construct that induces highly oriented myotube formation in myoblast cultures. MEW enables production of highly porous fibrous scaffolds with a wide range of fiber sizes and architectures. The objectives of this study were to determine the effects of MEW scaffolds and collagen concentration on construct contraction and quantify cellular viability over time. Our hypotheses were that higher collagen concentration reduces cell-mediated hydrogel contraction and that microfiber reinforcement enables maintenance of composite construct dimensional stability by preventing hydrogel contraction.

Materials and

Methods:  C2C12 myoblasts were seeded by resuspension in type I collagen solution. The seeding density used in both sets of experiments was 400,000 cells per mL of hydrogel. Contraction was determined with hydrogel-only cultures with collagen concentrations of 1.5, 2.0, and 3.0 mg/mL. Hydrogel-only cultures used 300 𝜇L of gel in each well while composite constructs used 100 𝜇L of hydrogel solution that polymerized on microfiber scaffolds. Composite constructs used a collagen concentration of 1.5 mg/ml and were cultured for 1 or 2 days in growth media. Microfiber scaffolds were printed using MEW processing of poly(ε-caprolactone) (PCL). Fibers with a 10μm diameter were layered to a height of 200 𝜇m. Three MEW scaffold designs were included: 1) aligned fibers without reinforcements (Aligned V3), 2) aligned fibers with orthogonal reinforcements (Aligned V0), and 3) non-aligned architecture (Isotropic). Control groups included 1) electrospun mesh (ES Mesh) and 2) tissue culture plastic (TCP). Cultures were stained with propidium iodide (PI) to determine dead cells and stained with DAPI after fixation for total nuclear count. Images were analyzed using ImageJ for the area overlap of PI and DAPI for each technical replicate (n=3 per biological replicate). Data are represented as mean ± standard error of the mean and analyzed by a two-way ANOVA (p < 0.05) using GraphPad Prism 10.

Results, Conclusions, and Discussions: Hydrogel contraction time was determined to decrease with increasing collagen concentration. The time at which hydrogel contracted from the well plate wall was 12 hours for 1.5 mg/mL, 24 hours for 2.0 mg/ml, and Day 6 for 3.0 mg/ml. Once gel-only controls started contracting and separating from wells, they rapidly contracted into a dense ball within 24 hours. However, the gel- composite constructs did not change in area over 8 days of culture. Viability of myoblasts seeded on MEW PCL scaffolds decreased on Day 2, whereas the two control groups of electrospun mesh and tissue culture plastic exhibited similar viability across Days 1 and 2. A two-way ANOVA revealed significant difference with day of culture (p=0.003), and no significant differences with scaffold design (p=0.0572), or interaction of time and scaffold design (p=0.0853).

Cell-mediated contraction of myoblast/Type-1 collagen hydrogel constructs is inhibited by higher collagen concentration and fully prevented over 8 days of culture by MEW fiber reinforcement. Since the concentration of collagen hydrogel can deleteriously affect engineered tissues, the use of MEW fiber reinforcement to maintain construct dimensional stability may be an advantageous strategy. Moreover, MEW fiber architecture can be used to direct cell orientation within 3D constructs via contact guidance cues. The viability of cells on the composite muscle constructs significantly decreased on day 2 compared with day 1. Longer term culture studies are ongoing to assess the viability and function of myoblasts within the composite constructs over time in culture.

Acknowledgements (Optional):