Wet-Spun Trojan Horse Cell Constructs for Engineering Muscle.

Anita F Quigley,Rhys Cornock,Robert M I Kapsa,Jeremy Crook,Javad Foroughi,Gordon G Wallace,Tharun Mysore,Joselito M Razal,Magdalena Kita,Simon E Moulton

doi:10.3389/fchem.2020.00018

Abstract

Engineering of 3D regenerative skeletal muscle tissue constructs (skMTCs) using hydrogels containing muscle precursor cells (MPCs) is of potential benefit for repairing Volumetric Muscle Loss (VML) arising from trauma (e.g., road/industrial accident, war injury) or for restoration of functional muscle mass in disease (e.g., Muscular Dystrophy, muscle atrophy). Additive Biofabrication (AdBiofab) technologies make possible fabrication of 3D regenerative skMTCs that can be tailored to specific delivery requirements of VML or functional muscle restoration. Whilst 3D printing is useful for printing constructs of many tissue types, the necessity of a balanced compromise between cell type, required construct size and material/fabrication process cyto-compatibility can make the choice of 3D printing a secondary alternative to other biofabrication methods such as wet-spinning. Alternatively, wet-spinning is more amenable to formation of fibers rather than (small) layered 3D-Printed constructs. This study describes the fabrication of biosynthetic alginate fibers containing MPCs and their use for delivery of dystrophin-expressing cells to dystrophic muscle in the mdx mouse model of Duchenne Muscular Dystrophy (DMD) compared to poly(DL-lactic-co-glycolic acid) copolymer (PLA:PLGA) topically-seeded with myoblasts. In addition, this study introduces a novel method by which to create 3D layered wet-spun alginate skMTCs for bulk mass delivery of MPCs to VML lesions. As such, this work introduces the concept of “Trojan Horse” Fiber MTCs (TH-fMTCs) and 3d Mesh-MTCs (TH-mMTCs) for delivery of regenerative MPCs to diseased and damaged muscle, respectively.

Highlights

In absence of effective pharmaceutical treatments for the long-term functional restoration of muscle tissue mass or function lost due to disease or trauma, numerous cell-based strategies have emerged to improve regenerative outcomes in damaged and dysfunctional skeletal muscle (Emery, 2002)
The fabrication process (Figures 1A,B) of PLGA (Figures 1D,E) and alginate (Figure 1F) Trojan Horse fibers containing myoblasts and subsequent biological viability of these fibers were optimized in vitro by analysis of process parameters: PLGA fibers were maximally over-grown with myoblasts immediately prior to further studies, whilst evaluation of alginate concentration and cell-loading parameters reproducibly yielded 1% and 2% alginate fibers containing large numbers of cells
There were clear signs that the alginate vehicle used to deliver the cells into the muscle environment had begun to undergo degradation even after 1 week, in stark contrast to poly(DL-lactic-co-glycolic acid) copolymer (PLA):PLGA/myoblast fibers, which were clearly evident as intact in recipient muscle after 12 weeks without cell survival and with significant foreign body response (Figure 3F). These results suggest that in vitro myoblast proliferation in, and migration from a biosynthetic alginate/cell construct with subsequent differentiation to muscle fibers within the surrounding environment shown in vitro in Figures 2I–K translate to cell behavior from within such constructs when implanted into muscle tissue in vivo

Summary

Introduction

In absence of effective pharmaceutical treatments for the long-term functional restoration of muscle tissue mass or function lost due to disease or trauma, numerous cell-based strategies have emerged to improve regenerative outcomes in damaged and dysfunctional skeletal muscle (Emery, 2002) These strategies include myoblast transfer therapies, growth factor delivery, stem cell delivery, gene therapy, and combinations of each (Emery, 2002; Mendell et al, 2010; Gombotz and Wee, 2012; Proto and Huard, 2013). The function of the SCs is to initiate (in the response to major injury) cell-based myoregeneration by proliferative differentiation of muscle precursors that eventually fuse into multi-nucleated myotubes, forming functional myofibres whilst retaining a myoregenerative muscle precursor cell (MPC) niche (Proto and Huard, 2013) In degenerative myopathies such as the muscular dystrophies, regenerative MPC loss occurs more rapidly than in nondystrophic muscle due to progressive myofibre degeneration and removal from the functional niche. In the case of volumetric muscle loss (VML) injury, the situation is dependent on cell delivery/matrix technology that allows for myoregenerative activity to be successfully localized to the VML lesion

Methods

Results

Conclusion