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Biomaterials

Biomaterials - Poster Session E

Poster N7 - Decellularized Extracellular Matrix with Hyaluronic Acid and Chondroitin Sulfate Helps Organize C2C12 Myoblasts Uniaxially Inside a 3D Printed Hollow Fiber

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Volumetric Muscle Loss (VML) results when 30% or more of total muscle mass is lost. This condition exceeds the regenerative capacity of remaining muscle and surgical interventions are needed. The current gold standard of treatment involving autologous muscle flap transfer is limited by graft tissue availability and donor site morbidity.

We propose the development of myoblast-seeded glycosaminoglycan-chitosan hollow fibers as a modular tissue engineering approach to forming transplantable muscle tissue. The hollow fibers are an insoluble ionic complex, made from the interaction of polycationic chitosan (CTS) and polyanionic glycosaminoglycans (GAG). We postulate that these hollow fibers will allow uniaxial organization of differentiating myoblasts. The candidate glycosaminoglycan (GAG) components include hyaluronan (HA) and chondroitin sulfate (CSA) and may be supplemented with collagen type I and/or decellularized muscle matrix (dECM).

In this study, we explored the effects of chitosan (CTS) flow rates and GAG composition on hollow fiber diameter. Additionally, previous experiments showed cells contracted the collagen in fibers, which led to cell aggregates instead of elongation. Since normal muscle growth is dependent on uniaxial orientation, we also investigated the effects of securing cell-seeded fibers on a support during culture.

Materials and

Methods:  Hollow fiber formation and cell encapsulation were accomplished simultaneously using a custom designed flow-focusing chip. C2C12 myoblasts (ATCC, Manassas, VA) were suspended in the GAG or GAG-ECM solution of interest (10 million cells/ml solution) and coaxially extruded with an external sheath of chitosan solution. Ionic interaction between the GAG and the coaxial chitosan flow generated an ionic complex membrane that formed the wall of the hollow fiber. Four GAG formulations were investigated: 1% Hyaluronan (HA), 1% HA + 1% CSA (HA-CSA), 1% HA + 1% CSA + 1.0 mg/ml decellularized muscle matrix (HA-CSA-dECM), and 1% HA + 1% CSA + 1.0 mg/ml collagen + 1.0 mg/ml dECM (HA-CSA-collagen-dECM). After formation, cell-seeded hollow fibers were surface stabilized by washing with 0.1 wt% polygalacturonic acid and normal saline before culturing in L-DMEM containing 10%FBS. Media was changed one hour afterwards to remove excess GAG. Calcein AM and DAPI were performed at day 2 and 5 of culture, to check for viability and location of cells. The 3D printed fiber support was printed using ANYCUBIC Photon 3D printer, UV Cured for 5 minutes, boiled in DI water at 100⁰C for 30 minutes, and kept in 70% Isopropanol for 24h to remove any uncured resin. Prior to culture, the support was kept in 70% Ethanol overnight then rinsed in sterile saline before the cell-seeded fibers were added on.

Results, Conclusions, and Discussions: We previously reported on a (Figure 1A) 3D-printed, microfluidic, flow focusing chip used to produce coaxially extruded GAG-chitosan hollow fibers. (Figure 1C) In this study, higher CTS flow rates decrease fiber diameter across all conditions, and addition of CSA to the GAG formulation decreases fiber diameter approximately by half. CSA-containing conditions show relatively similar fiber diameters, with 1%HA, 1%CSA yielding lowest fiber diameter on average, a formulation which when combined with either collagen or dECM, or both, can provide proper cell support.

In previous experiments, (Figure 2A) myoblasts seeded inside HA-Collagen fibers were not elongating. We hypothesized that was caused by the cells contracting the collagen inside the free-floating fibers. We tested this theory by comparing (Figure 2C) a set of HA-CSA-Collagen fibers free-floating, versus (Figure 2E) HA-CSA-Collagen (Figure 1B) fibers on a 3D printed support. (Figure 2E) Under constant tension, the cells orient themselves uniaxially, while cells that are free-floating aggregate. This supports our hypothesis that cells which are in free-floating fibers, contract the collagen and fiber and therefore are not able to organize themselves uniaxially as they do when they under constant tension on the support.

In comparing the four proposed GAG formulations, (Figure 3A) cells in the HA-CSA condition appear round throughout the entire fiber, whereas (Figure 3B) cells in the collagen condition show cell elongation. (Figure 3C) In the HA-CSA-dECM and (Figure 3D) HA-CSA-Collagen-dECM, the cells organize themselves uniaxially. These results suggest that collagen organizes within the fiber over long distances due to gel formation, and the decellularized matrix does not organize over long distances. Therefore, the cells can contract and pull on the collagen and this causes fiber contraction unless the fibers are immobilized on the support. In the case of the dECM, cells appear to exhibit less aggregation and a more uniform distribution.

Current studies are exploring the effects the addition of dECM has on detailed morphology and expression of myoblast markers. Future studies will explore culture of primary myoblasts in HA-collagen-dECM hollow fibers versus the remaining three conditions. We will report on cell differentiation, myotube formation and orientation, and presence of myotube contraction.

Acknowledgements (Optional):