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Muscle-Specific ECM Derived Hydrogel For 3D Printing Facilitates Differentiation Of Adipose Derived Stem Cells Into Muscle Cells
Solmaz N. Niknam-Bienia, MD, MHA; Alexandra L. Rutz, MS; Sumanas W. Jordan, MD, PhD; Ian Chow, BS; Thomas A. Mustoe, MD; Ramille N. Shah, PhD; Seok Jong Hong, PhD; Robert D. Galiano, MD
Northwestern University, Chicago, IL, USA.
Significant skeletal muscle defects can be difficult to regenerate as speedy repair is favored and therefore fibrosis predominates leading to muscle deinnervation and atrophy. Despite substantial advances in biomaterials, the challenge remains the development of a new biomaterial that can coax stem cells to differentiate into muscle and thus serve as a scaffold for potential regeneration. In this study, we propose a muscle-specific extracellular matrix (M-ECM) derived hydrogel for 3D printing that we hypothesize improves differentiation of adipose derived stem cells (ADSC) into muscle cells, improves cell alignment, and demonstrates synchronized intercellular electroconduction. Microfibers of M-ECM were explored for the potential of generating aligned muscle fibers for the ultimate goal of engineering muscle implants.
ADSCs were isolated from rats, cultured, and differentiated into osteocytes, chondrocytes, and adipocytes to ensure multipotency. M-ECM was processed into a hydrogel suitable for extrusion biofabrication from decellularized rat abdominal wall muscles. ADSCs were plated with muscle differentiation media in 3 groups: onto a standard culture plate, onto a collagen control, and on a M-ECM hydrogel. Samples were analyzed via immunohistochemistry and immunofluorescence using antibodies against Pax7, Myf5, myogenin, MyoD, desmin, and skeletal muscle specific actin. Hydrogel scaffolds of collagen and M-ECM seeded with GFP labeled ADSCs were implanted into a critical size defect on the latissimus dorsi of the rat. A third group consisting of a critical size defect alone was also included. The samples were harvested at 1, 3, 10, and 20 days to again be analyzed for muscle growth. Collagen and M-ECM hydrogels were then extruded into fibers and analyzed using the same antibodies. Contractile forces were also investigated by applying electrical stimulation across the construct and measuring contraction distance.
The M-ECM derived hydrogel showed improved differentiation of the ADSCs compared to the collagen and standard culture plates in vitro as evidenced by an increased number of further differentiated cells under immunohistochemistry and immunofluorescence. The M-ECM derived hydrogel microfibers demonstrated superior alignment of differentiated muscle cells. Contractile properties of M-ECM derived hydrogel fibers were superior to collagen fiber controls observed by an increased distance of contraction.
Muscle-specific ECM derived hydrogel provides specific differentiation cues and retains bioactivity thus facilitating ADSCs to differentiate into muscle cells. M-ECM can be extruded to form microstrands signifying the ability to fabricate a single muscle fiber and to be utilized for 3D printing. 3D printing will enable direct control over microstrand placement to build scaffolds with defined microarchitecture that is vital to regeneration of highly organized skeletal muscle. These findings unlock an exciting opportunity for further development of 3D printed biomaterials and regeneration of functional skeletal muscle from stem cells and progenitor cells other than muscle.
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