(337c) Aligned and Conductive 3D Collagen Scaffolds for Skeletal Muscle Tissue Engineering

Introduction: Bioelectrical stimuli are important in organ regeneration and are integral to normal skeletal muscle function, including the cell-cell signaling that coordinates synchronous contraction [1]. Current biomaterial approaches to simulate the endogenous conductivity of tissue often employ conductive polymers such as polypyrrole, polyaniline, and poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) in two-dimensional (2D) films or fibers [2]. While these approaches have demonstrated beneficial effects on myoblast differentiation and maturation, the architecture of these materials fails to mimic the three-dimensional (3D) anisotropic organization of native muscle tissue [3]. Our work describes the development of a 3D conductive collagen scaffold with a highly aligned, anisotropic pore structure for muscle tissue engineering.

Results: Conductive aminated-polypyrrole (APPy) powder doped with FeCl₃was sieved to a diameter of ~ 44 µm and homogenously distributed into a suspension of bovine collagen and chondroitin sulfate in acetic acid. Anisotropic scaffolds were then prepared using an ice templating approach where collagen-APPy suspension was added to a custom-designed mold to promote directional heat transfer during lyophilization [4]. The APPy content of the scaffold was stabilized post-lyophilization by covalently crosslinking the carboxylic acids on the collagen backbone to primary amines of the APPy using EDC/NHS chemistry. The resulting scaffolds contained a highly aligned 3D pore microstructure with an average pore size of 185 ± 47 µm. Conductivity of the scaffolds was modulated by incorporating varying amounts of APPy powder into the starting collagen suspension. The addition of 0.06 wt% APPy resulted in a roughly two-fold increase in conductivity (0.38 ± 0.11 S/cm) when compared to a collagen scaffold control (0.20 ± 0.03 S/cm). Additionally, APPy-doped collagen scaffolds displayed a similar linear viscoelastic (LVE) region when compared to collagen controls (LVE limit of ~ 1% strain) and a comparable storage modulus of ~ 10 kPa, similar to moduli previously shown to be beneficial for muscle tissue engineering [5,6]. C2C12 mouse myoblasts cultured over one week within APPy-modified collagen scaffolds showed a 42% increase in metabolic activity on day 7 compared to day 0. This was not statistically different when compared to the collagen control. Furthermore, F-actin and collagen staining after 7 days of culture on APPy-collagen scaffolds indicated improved cell alignment and cytoskeletal organization within the scaffold microstructure with increasing levels of APPy incorporation.

Conclusions: We developed a highly aligned, 3D, conductive collagen scaffold by directional lyophilization of an APPy-doped collagen suspension for skeletal muscle tissue engineering. Increasing conductivity of the scaffold facilitated improved cell alignment and cytoskeletal organization along the scaffold pore structure in a manner reminiscent of healthy muscle. Moreover, the addition of APPy did not detrimentally alter the mechanics of the scaffold nor reduce C2C12 mouse myoblast viability. Ongoing work is exploring the differentiation and maturation of C2C12 cells in these scaffolds.

References: 1) Breukers R et al., Journal of Biomedical Materials Research, 2010, 93: 256-268; 2) Balint R et al., Acta Biomaterialia, 2014, 10: 2341-2353; 3) Gilles A et al., Muscle Nerve, 2011, 44: 318-331; 4) Caliari S et al., Biomaterials, 2011 32: 5330-5340; 5) Engler A et al., Cell, 2006 126: 677-689; 6) Gilbert P et al., Science, 2010 329: 1078-1081