An Injectable Decellularized Muscle Matrix for Skeletal
Muscle Tissue Engineering and its Mitogenic Properties
Jessica A. DeQuach1, Joy E. Lin1, Cynthia Cam1, Diane Hu1 and Karen L. Christman1
1
Department of Bioengineering, University of California, San Diego, La Jolla, CA 92092-0412
Tel: 858-534-9628 E-mail: christman@bioeng.ucsd.edu
Summary: Decellularized skeletal muscle matrix is able to self-assemble to form a threedimensional scaffold in situ and when injected into a hindlimb ischemia model is able to promote
increased neovascularization and muscle cell recruitment when compared to injectable collagen
scaffolds. We have additionally studied in vitro effects of the skeletal muscle matrix as a mitogen
for cellular proliferation of skeletal myoblasts.
Skeletal muscle injury and other intrinsic myopathies may lead to significant damage that prevents
complete regeneration of function. The current clinical approach is to use autologous tissue transfer, or
muscle flaps, however the tissue frequently does not transplant well. Using tissue engineering approaches,
scaffolds can be placed at the injury site to replace the biological tissue through native cell recruitment.
However, many scaffolds do not contain the same complex components that are found in the native tissue,
and may be difficult to store and transport. Thus, we have developed a liquid form of skeletal muscle
matrix that is derived from decellularized porcine leg muscle, which can be injected to form a fibrous and
porous scaffold to fill a defect and may be an off-the-shelf treatment. Our objective was to assess whether
the skeletal muscle matrix acts as a mitogen for cellular proliferation of skeletal myoblasts in vitro and to
use this material in a hindlimb wound rat model to determine the regenerative effects over time.
Skeletal muscle matrix was extracted from porcine leg, and decellularized using detergents. The matrix was
then lyophilized and milled to form a powder that was then digested enzymatically to form liquid matrix,
further processing by re-lyophilizing the sample allows for long-term storage so that water can be added at
a later time to resuspend at a physiological pH. Mass spectroscopy was used to determine protein content
and found many collagen types, other fibrous proteins and proteoglycans, and a Blyscan assay detected
glycosaminoglycans. Thus, our processed matrix retains components found in native skeletal muscle.
Femoral artery resection to create a hind limb wound region in Sprague Dawley rats. One week post injury,
150 μL of 6 mg/ml skeletal muscle matrix or 6 mg/ml of collagen I were injected into the location of
muscle injury. Leg muscle was collected, sectioned and stained with H&E to determine major tissue
morphology. Immunohistochemistry was used to quantify arteriole formation and muscle cell recruitment.
Results demonstrated that arteriole density was greater in the skeletal muscle matrix injection region when
compared to collagen. Additionally, more proliferating muscle cells were recruited to the skeletal muscle
matrix scaffolds in comparison to the collagen scaffold.
Mitogenic properties of skeletal muscle matrix was assessed via dosing the culture medium of C2C12 with
skeletal muscle matrix, collagen or pepsin and compared to cells cultured without additives. A picogreen
assay was used to quantify double stranded DNA content, which should correlate to cell proliferation. The
skeletal muscle matrix, when added into the media demonstrates a mitogenic effect on skeletal myoblasts
with a 1.42 fold increase in proliferation compared to growth media (p = 0.03) and a 1.67 fold increase
compared with collagen (p = 0.0006) at day 7. Thus we demonstrate that the decellularized skeletal muscle
matrix is able to form a scaffold in situ that recruits arteriole formation and muscle cell infiltration, and the
matrix fragments are able to affect proliferation of muscle cells in vitro as a mitogenic factor.