Engineering 3D skeletal muscle primed for neuromuscular regeneration following volumetric muscle loss
Introduction
The repair mechanism for skeletal muscle is severely compromised following volumetric muscle loss (VML), thus leading to chronic functional deficits [1,2]. Current treatments are often unsuccessful in restoring muscle function and are limited by donor site morbidity, lack of donor tissue, and the need for highly skilled surgical teams [3]. It was recently demonstrated that VML is accompanied by significant motoneuron axotomy and lost interaction between neurons and the injured skeletal muscle, a probable cause for the heightened levels of lost muscle function seen post-VML [4]. Tissue engineered grafts have great potential to create clinical treatment options for regeneration of muscle with VML, but previous approaches to regenerate the injured muscle remain limited in their ability to encourage neuromuscular junction (NMJ) regeneration within the healing tissue. Engineered muscle constructs that incorporate methods to encourage neural infiltration and the formation of functional NMJs post-VML provide promising avenues to restore interaction between the muscle and nerve.
Prior strategies to promote NMJ regeneration post-VML have included neurotization [5,6], the use of rehabilitative exercise [[7], [8], [9], [10]], and implantation of a pre-innervated construct [11]. These approaches have moderately increased neural infiltration, force recovery, and NMJ formation within and around the defect site. Despite incremental neuromuscular regeneration, the nerve and NMJ densities within the defects remain low and the newly formed NMJs exhibit abnormal morphologies. In addition, there has been little effort to quantify neuromuscular regeneration across the entire defect area, instead relying on subsets of the defect region at high magnifications that likely do not accurately represent variability in expression across the defect region. In native tissues, the mature NMJ contains densely clustered postsynaptic acetylcholine receptors (AChR) optimized for efficient signal transfer across the neuromuscular synapse and effective muscle contraction [12]. Agrin, a large heparan sulfate proteoglycan secreted by the nerve terminal, is vital for AChR cluster stabilization during embryonic development [13] and has been utilized extensively to induce AChR clustering in cultured myotubes [[14], [15], [16], [17], [18], [19], [20], [21], [22]]. Muscle constructs containing agrin physically mixed into a fibrin hydrogel and seeded with mouse C2C12 myoblasts were implanted subcutaneously in a non-VML defect near the peroneal nerve for 8 weeks and resulted in increased nerve infiltration, NMJ formation, and vascular infiltration [14], demonstrating the potential neuromuscular therapeutic benefits of scaffold-mediated agrin delivery in vivo. In this study, we resolved to test whether the delivery of soluble or chemically-tethered agrin could promote improved NMJ regeneration in the treatment of VML defects.
Although release of bioactive molecules from engineered constructs has been utilized in VML treatment to promote vascular infiltration [[23], [24], [25], [26]] and myogenesis [[25], [26], [27]], there have been no previously published constructs used for VML treatment that have incorporated a bioactive factor to induce nerve infiltration and the formation of neuromuscular junctions. In addition, prior VML treatments with scaffold-mediated delivery of bioactive molecules have utilized physical entrapment or passive adsorption to the scaffold prior to implantation, which rely on diffusion of the bioactive agent to the surrounding tissues [28]. In contrast, scaffolds incorporating immobilized bioactive agents offer the ability to control spatiotemporal presentation and local signaling to the regenerating tissue, avoid poor targeting efficiency, and extend factor bioactivity over time following implantation [[29], [30], [31]]. Our group has previously utilized electrospun fibrin microfiber bundles that mimic the native properties of skeletal muscle combined with C2C12 myoblasts to promote functional and histological regeneration post-VML [32]. In the current study, we have enhanced our in vitro muscle constructs to promote neuromuscular regeneration in VML defects through the delivery of agrin. We tested the hypothesis that immobilized agrin tethered to the surface of C2C12 myoblast-seeded microfiber bundles would maintain its in vitro bioactivity and induce AChR clustering in myotubes cultured on the 3D constructs. In addition, we tested the ability of tethered agrin constructs implanted in a murine VML defect model to improve neuromuscular regeneration via recruitment of acetylcholine receptors to overlap with nerves and form neuromuscular junctions at 4 weeks compared to constructs pre-treated with soluble or zero agrin.
Section snippets
Electrospinning fibrin scaffolds
Fibrin scaffolds were electrospun in a sterile environment with sterile solutions as previously described [32,33]. Parallel syringes containing solutions of fibrinogen (Sigma) or sodium alginate (Sigma) were connected via a Y-connector and extruded by syringe pumps with an applied voltage of 3–5 kV applied to a 27-G needle tip utilizing 1% fibrinogen with an extrusion rate of 4 ml/h and 0.75% alginate with an extrusion rate of 1 ml/h. Poly(ethylene oxide) (PEO, average Mv ~ 4000 kDa, Sigma) was
AChR cluster 3D spatial distribution is determined by agrin delivery method
Electrospun fibrin hydrogels designed to mimic the hierarchical structure and alignment of skeletal muscle were fabricated as previously described. A protocol to chemically tether proteins to the electrospun fibrin scaffolds using the EDC zero-length crosslinker was initially developed and validated using a fluorescent Cy3 antibody which has a similar size to agrin (Fig. S1A). Agrin was chemically tethered to acellular scaffolds at 0.08–2 μg/scaffold prior to seeding with C2C12 myoblasts, after
Discussion
Successful regeneration of VML defects includes the formation of mature NMJs that allow for efficient neural signal propagation and subsequent muscle contraction. Significant advances in the field of skeletal muscle tissue engineering have demonstrated varied levels of muscle tissue regeneration and functional recovery post-VML [9,23,[38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48]], but remain limited in their ability to promote neuromuscular regeneration. To date, there have
Conclusions
In conclusion, engineered muscle constructs pre-treated with agrin form dense AChR clusters in vitro and promote neuromuscular regeneration upon implantation in VML defects for four weeks. While all treatment groups resulted in improved muscle function, constructs pre-treated with both soluble and tethered agrin caused increased neurofilament and blood vessel infiltration, NMJ formation, and the presence of eMHC + regenerating myofibers compared to no agrin controls. In addition, sustained in
Author contributions
Jordana Gilbert-Honick: Designed research, Performed research, Analyzed data, Wrote the paper, Shama R. Iyer: Performed research, Sarah M. Somers: Performed research, Analyzed data, Hannah Takasuka: Performed research, Richard M. Lovering: Designed research, Analyzed data, Kathryn R. Wagner: Designed research, Analyzed data, Hai-Quan Mao: Designed research, Analyzed data, Warren L. Grayson: Designed research, Analyzed data, Wrote the paper.
Data availability
The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations.
Declaration of competing interest
The authors declare no conflict of interest.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
Funding was provided by the Maryland Stem Cell Research Fund (2016-MSCRFI-2692), the Wilmer Core Grant for Vision Research, Microscopy and Imaging Core Module (EY001765), the NIH (NIAMS NRSA F31 AR071759 and K01 AR074048), and the MDA DG 577897.
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