Biofabricating murine and human myo-substitutes for rapid volumetric muscle loss restoration.

Marco Costantini,Bert Blaauw,Dror Seliktar,Stefano Testa,Jacopo Baldi,Gianni Cesareni,Carmine Zoccali,Luisa Castagnoli,Minghao Nie,Alberto Rainer,Ersilia Fornetti,Cesare Gargioli,Piotr Garstecki,Libero Vitiello,Stefano Cannata,Sergio Bernardini,Wojciech Święszkowski,Claudia Fuoco,Carles Sánchez Riera ,R Biagini

doi:10.15252/emmm.202012778

Abstract

The importance of skeletal muscle tissue is undoubted being the controller of several vital functions including respiration and all voluntary locomotion activities. However, its regenerative capability is limited and significant tissue loss often leads to a chronic pathologic condition known as volumetric muscle loss. Here, we propose a biofabrication approach to rapidly restore skeletal muscle mass, 3D histoarchitecture, and functionality. By recapitulating muscle anisotropic organization at the microscale level, we demonstrate to efficiently guide cell differentiation and myobundle formation both in vitro and in vivo. Of note, upon implantation, the biofabricated myo‐substitutes support the formation of new blood vessels and neuromuscular junctions—pivotal aspects for cell survival and muscle contractile functionalities—together with an advanced muscle mass and force recovery. Altogether, these data represent a solid base for further testing the myo‐substitutes in large animal size and a promising platform to be eventually translated into clinical scenarios.

Highlights

The importance of skeletal muscle tissue is undoubted being the controller of several vital functions including respiration and all voluntary locomotion activities
We presented an extrusion-based 3D bioprinting approach that enables to biofabricate hydrogel scaffolds composed of aligned myoblast-laden fibers (Costantini et al, 2017)
Thanks to the bio-technological progress of the last two decades, substantial advances in the tissue engineering field have been achieved with the fabrication of liver tissue with sinusoids (Toh et al, 2009; Schu€tte et al, 2011), lung functions (Huh et al, 2010), spleen (Baker, 2011), skeletal muscle model functionality (Nagamine et al, 2011; Serena et al, 2016), in vitro models that are capable of mimicking native organ morphology and functionalities

Summary

Introduction

The importance of skeletal muscle tissue is undoubted being the controller of several vital functions including respiration and all voluntary locomotion activities. Despite the introduction of new technologies and refined protocols for culturing engineered muscles—derived from either murine or human muscle progenitors—so far, SM maturation and maintenance have been only partially recapitulated in vitro and, most importantly, to a small scale (Leng et al, 2012; Zhang et al, 2015) These approaches, while promising, are still far from satisfactorily addressing the second key challenge of SMTE—i.e., the development of macroscopic tissue equivalents of a size suitable for treating volumetric muscle loss (VML): a condition resulting from large traumatic injury or massive surgical ablation upon tumor removal (Passipieri & Christ, 2016; Gilbert-Honick & Grayson, 2019). Our findings demonstrate that by optimizing key parameters such as: (i) hydrogel precursor composition (i.e., bioink), (ii) cell seeding density, (iii) fiber architecture, and (iv) culturing protocol, we are able to successfully fabricate advanced artificial myo-substitutes that can be used to restore SM mass loss and functionality in vivo

Results

Discussion

Materials and Methods

Conflict of interest