Speaker
Description
Introduction
The functionality of skeletal muscle tissue (SMT) hinges on its hierarchical anisotropic microstructure. Conventional 2D cell cultures are poorly biomimetic and unable to properly support in vitro engineering of SMT, triggering the research towards more reliable and predictive in vitro models [1]. Biomimetic fiber-based scaffolds provide topographical cues supporting SMT in vitro engineering, but optimal biomaterials, methods and technologies for a controlled 3D fiber organization are missing. In the biofabrication field, conventional bioinks lack the multiscale anisotropy and the structural fidelity required to guide muscle fiber alignment and maturation. This work addressed such limitations by designing 3D in vitro models of SMT using natural polymers and advanced fabrication techniques.
Methods
Next-generation fibrous bioinks, composed of fragmented, electrospun gelatin fibers (f) uniformly embedded in an alginate/gelatin hydrogel matrix (f-ALG/Gel) were designed. The hydrogel was crosslinked using calcium ions and microbial tranglutaminase. Bioink concentration and composition was optimized through accurate rheological and printability characterization as well as in vitro cell adhesion tests using C2C12 murine myoblasts. The effect of the anisotropic microenvironmental cuesof bioprinted structures on C2C12 cell morphology, phenotype and directional growth was quantified by immunofluorescence analysis. The pipeline for image analysis employed Gaussian filtering and CLAHE to enhance contrast and reduce noise while preserving edge information. Initial segmentation was performed using global thresholding, followed by morphological operations to refine detected structures. A watershed-based algorithm was applied to separate overlapping features, ensuring accurate identification of nuclei and fibers.
Results
Upon extrusion-based 3D bioprinting, shear-induced alignment of f-GFs enabled the fabrication of microfilament-based scaffolds with intrinsic uniaxial anisotropy. The resulting constructs exhibited high shape fidelity, viscoelastic properties, and physiologically relevant stiffness (Young’s modulus: 16.1 ± 1.7 kPa). In vitro studies using C2C12 murine myoblasts demonstrated that the embedded f-GFs provided strong topographical guidance, enhancing cell alignment and myogenesis. After 14 days culture, the f-ALG/Gel scaffolds supported a 2.5-fold increase in myotube fusion index and length, alongside reduced angular dispersion, compared to control bioinks. These effects were achieved without the need for biochemical stimulation of cell differentiation through a specific culture medium, underscoring the key role of hierarchical structural cues at the micro- and nanoscale on C2C12 differentiation and maturation.
Discussion
This work proposed a scalable, cell-compatible strategy to recapitulate the hierarchical organization of SMT through 3D bioprinted constructs, offering a new class of structurally instructive bioinks. In the future, the substitution of C2C12 cells with primary human myoblasts or myogenic cells derived from hiPSCs will allow the engineering of human-relevant SMT.
The platform holds broad potential for applications in regenerative medicine, skeletal muscle tissue modelling and the engineering of cultured meat.
References
[1] Zhuang, P. et al.; 193, 108794 (2020).
Acknowledgement
This work was supported by:
the European Research Council (ERC) under the European Union's Horizon 2020 Research and Innovation Programme, through BIORECAR ERC Consolidator project (Grant Agreement No. 772168).
- the ERC under the European Union's Research and Innovation Programme through ERC-2023-POC EMPATIC project (Grant Agreement No 101158332).
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