Extruded alginate tubes with myogenic potential

Abstract

Background: There are currently no proven methods to reverse muscle loss in humans, which is caused by trauma (e.g., volumetric muscle loss, VML), genetic neuromuscular diseases (e.g., muscular dystrophies, MDs), and accelerated senescence (e.g., sarcopenia). Since muscle tissue is capable of regeneration through muscle satellite cells (MuSCs), the implantation of autologous (or other) donor MuSCs and MuSC-derived myoblasts into host muscles can promote donor-cell-derived myogenesis. Direct injection or implantation of MuSCs or MuSC-derived myoblasts into host muscles only promotes minimal donor-cell-derived myogenesis, whereas implantation of MuSCs/myoblasts along with associated muscle tissue (muscle fibers, extracellular matrix, neurovascular pathways, etc.) gives better results.

Methods: We aim to leverage the benefits of constraining donor myogenic cells within a template that resembles muscle tissue. In this paper, we present a workflow for basic and translational studies aimed at promoting donor-cell-derived myogenesis to increase functional muscle mass in mice. Our workflow involves preparing a slurry of 10% sodium alginate mixed with myogenic cells in cell culture media, extruding the cell-containing slurry into 10% calcium lactate to form tubes, and implanting the cellularized alginate tubes into host muscle.

Results: Our data suggest that, the extruded alginate tubes can tolerate a peak stress of 1892 ± 527 mN, that the elastic range is at ~75-125% strain beyond initial length, and that the Young's modulus (stiffness) is 14.17 ± 1.68 %/mm². Importantly, these mechanical properties render the alginate tubes suitable for a published technique known as minimally-invasive muscle embedding (MIME) that was developed by us to implant myogenic material into host muscle. MIME involves threading donor myogenic tissue into a needle track created within a host muscle. Cellularized alginate tubes implanted into the tibialis anterior muscle of previously euthanized mice had numerous hematoxylin-stained structures similar to nuclear staining, supporting the idea that our alginate tubes can support cell seeding. Alginate tubes that were seeded with MuSCs, incubated in MuSC/myoblast growth (i.e., proliferation) media for two days, incubated in myotube differentiation media for six days, and then minced and reseeded in new dishes, were able to promote in vitro myoblast outgrowth over several days.

Discussion: This pilot study is limited in its translational scope because it was performed in vitro and with previously euthanized mice. Additional studies are needed to confirm that cellularized alginate tubes can promote the de novo development of donor-cell-derived muscle fibers, which can contribute to contractile force production.

Conclusion: Alginate tubes with MuSC/myoblasts can be generated by a simple extrusion method. The alginate tubes have sufficient mechanical strength to tolerate insertion into a host muscle, in a minimally-invasive manner, through a needle track. The cellularized alginate tubes demonstrate myogenic potential since they are capable of being maintained in culture conditions for several days, after which they can still facilitate myoblast outgrowth in a dish.

Figure 1.. Alginate tube mechanical testing

We have developed the technology to generate cell-encapsulated alginate tubes (A). Briefly, MuSCs harvested from donor muscle tissue are combined with media to give a 10% sodium alginate slurry. The slurry is fed into a syringe and extruded into Ca-lactate through a 16-gauge needle with an automatic syringe pump. Mechanical testing with a force measurement system suggests that the alginate tubes can withstand up to 1500 mN and can undergo strain >150% of its initial length (B, C).

Figure 2.. Alginate tubes implanted into the mouse TA muscle by MIME

Alginate (10% sodium alginate) tubes fabricated by a simple extrusion process are amenable to mechanical handling required for implantation into host muscle with the MIME technique (A, B). Frozen, 20 µm cross sections that were stained with hematoxylin and eosin show that the alginate tubes implanted by MIME are within the compartment of the TA muscle (C, D). Cellularized alginate shows evidence of nuclear staining with hematoxylin, whereas alginate that has not been cellularized does not show nuclear staining.

Figure 3.. Myoblast outgrowth from minced alginate tubes

Alginate tubes that were cellularized and maintain in cell culture for several days (two days in growth media and six days in differentiation media) were minced and reseeded in 60 mm plates with growth media. The purpose of this experiment was to test if cell-seeded alginate tubes can promote myoblast outgrowth as documented with actual muscle tissue. Our data suggest that over several days after reseeding, minced alginate tubes are able to facilitate myoblast outgrowth as evidenced by increasing confluence over time.