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3D bioprinting directly affects proteomic signature and myogenic maturation in muscle pericytes-derived human myo-substitute

ABSTRACT: Skeletal muscle tissue engineering (SMTE) has recently emerged to address major clinical challenges such as volumetric muscle loss. Here, we report a rotary wet-spinning (RoWS) biofabrication technique for producing human myo-substitutes with biomimetic architectures and functions. Here, we demonstrate how the proposed technique may be used to establish a well-tailored, anisotropic microenvironment that promotes exceptional myogenic differentiation of human skeletal muscle-derived pericytes (hPeri). Using high-resolution mass spectrometry-based proteomics with the integration of literature-derived signaling networks, we uncovered that i) 3D biomimetic matrix environment (PEG-Fibrinogen) confers a lower mitogenicity microenvironment compared to standard 2D cultures, favoring the formation of contractile-competent bundles of pericytes-derived myotubes in an anchoring-independent 3D state, and ii) the bioprinting method promotes an upregulation of muscle matrix structural protein besides increasing contractile machinery proteins with respect to 3D bulk cultures. Finally, in vivo investigations demonstrate that the 3D bioprinted myo-substitute is fully compatible with the host ablated muscular tissue, exhibiting myo-substitute engraftment and muscle regeneration in a mouse VML model. Overall, the results show that 3D bioprinting has a superior capability for controlling the myogenic differentiation process on a macroscale and, with future refining, may have the potential to be translated into clinical practice.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Alessio Reggio

LAB HEAD: Cesare Gargioli

PROVIDER: PXD043605 | Pride | 2025-10-20

REPOSITORIES: Pride

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			Action	DRS
	2022-07-04_MS_CG_m2D_1.raw	Raw
	2022-07-04_MS_CG_m2D_2.raw	Raw
	2022-07-04_MS_CG_m2D_3.raw	Raw
	2022-07-04_MS_CG_m2D_4.raw	Raw
	2022-07-04_MS_CG_m3D_1.raw	Raw

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Project description:Background Three-dimensional (3D) bioengineered models of human skeletal muscle are a promising approach for studying muscle development, function and disease in vitro. These models more closely resemble the complexity of native muscle tissue than two-dimensional (2D) monolayer culture and allow for functional measurements to be performed. However, a more complete understanding of how culture condition and duration impacts the myotube maturity and function is required to validate the transition from 2D to 3D culture of muscle cells. Methods Human skeletal muscle cells were cultured as either 2D monolayers or within 3D fibrin-based hydrogels as muscle constructs for up to 21 days. Quantitative proteomic analysis and functional assessments, including contractile force and cross-sectional area measurements, were conducted to evaluate the impact of culture conditions and duration on muscle cell differentiation. Results Proteomic analysis revealed myoblasts differentiated into myotubes by 8 days of differentiation in both 2D and 3D environments. However, the proteomic profiles of myotubes varied significantly between the two culture environments. At day 8 of differentiation, myosin heavy chain isotype abundance indicated a predominantly slow-twitch phenotype in 3D constructs, compared to a mixed fibre type phenotype in 2D monolayers. By day 21 of differentiation, 3D muscle constructs displayed improved mitochondrial maturity, extracellular matrix remodelling, and signs of transitioning towards a fast-twitch phenotype. This prolonged culture duration also resulted in increased passive tension but decreased peak contractile force in 3D muscle constructs. Conclusions This study demonstrates that 3D culture promotes maturity in human skeletal muscle cells, mimicking the biochemical cues and energy demands seen in native muscle tissue. The data highlights the importance of selecting appropriate culture conditions and durations when studying skeletal muscle cells in vitro and suggests 8 days of differentiation as optimal for achieving peak contractile force in 3D muscle constructs.

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3D bioprinting directly affects proteomic signature and myogenic maturation in muscle pericytes-derived human myo-substitute

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