The present study describes a decellularization method for intact skeletal muscle without the use of detergents or proteolytic enzymes. The method utilized actin disruption by treatment with latrunculin B, cell lysis by osmotic shock, myosin depolymerization by exposure to high ionic strength salt solution, and DNase I treatment to remove residual DNA from skeletal muscle. This method successfully removed muscle fibers and degraded sarcomeric components in the muscle tissue without altering the ECM structure or mechanical properties.
Since DNA quantification indicated the presence of a small amount of DNA in the decellularized tissue, tissue sections were stained for satellite cells that may be more resistant to the decellularization protocol. Negative Pax7 (satellite cells) and DAPI staining indicated that any DNA remaining in the decellularized muscles was degraded and not contained within intact cell nuclei.
An approximately 40% reduction in the GAG content of the decellularized tissue was observed, which could be due to removal of GAGs associated with the cell membrane; almost 30% of GAGs are associated with the cell membrane,30
and these GAGs may have been removed with the sarcolemma during the decellularization process. Decellularization methods using trypsin and Triton X-100 remove GAGs from skeletal muscle,21
but this effect appears to be tissue dependent since the use of trypsin and/or Triton X-100 on other tissues has yielded disparate results.31–33
Collagen content was unchanged in decellularized muscle, showing that this method did not remove the most abundant ECM structural component. Together, these results provide evidence that the decellularization method described here is minimally disruptive to native ECM. Further, comparison with the method of Stern et al.
showed that our decellularization method was more efficient at removing DNA and retaining GAGs in intact muscle compared with a method that uses a proteolytic enzyme (trypsin) and a detergent (Triton X-100).21
SEM images from decellularized muscles () suggested that muscle fibers were removed from the tissue, but that the overall architecture of the ECM was maintained when compared with the untreated tissue. SEM images of skeletal muscle endomysium obtained by Trotter and Purslow show a thinner, more fibrous matrix than we obtained in this study.34,35
The differences in the endomysial thickness could be attributed to the different sample preparation methods. Totter et al.
treated muscles with NaOH, which removed the sarcolemma, basement membrane, and any GAGs associated with the endomysium, leaving only collagen fibrils within the ECM.34
The thicker ECM observed in our decellularized tissues is likely due to the presence of GAGs and the basement membrane.
Mechanical analysis of the decellularized muscles confirmed the mechanical integrity of the decellularized tissue since no difference was observed between the untreated and decellularized muscle bundle stress–strain relationship (). These results provide support for the concept that the ECM is the primary passive load bearing structure in skeletal muscle, not the fibers themselves. This result must be tempered by the fact that there was no unambiguous reference length available for determination of the stress–strain relation for decellularized muscle bundles. However, future studies can potentially overcome this limitation by referencing strain to sarcomere length before decellularization or by measuring the relationship between collagen crimp pattern and tissue strain.
The ability of the decellularized muscles to support adhesion and survival of C2C12 cells indicates their cytocompatibility, which is in agreement with other studies.4,5,21
The use of decellularized muscle has clinical applications in repair of severe skeletal muscle injuries where the muscles cannot undergo complete regeneration. Detergent-decellularized muscle has been used as a scaffold to repair muscle defects in animal models4,5,18
; however, complete functional regeneration has not yet been shown. Biochemical and mechanical signals of the ECM play a key role in the activation of muscle progenitor cells and their differentiation,11,21,36
and thus the use of muscle ECM that contains these signals may be more effective at achieving functional regeneration of impaired muscles. This decellularization method may be used to study the changes that occur in muscle ECM with various myopathies and may serve as a diagnostic tool by identifying characteristic patterns in muscle ECM composition or architecture. In addition, tissues decellularized by this method may be used to measure directly the passive mechanical properties of skeletal muscle ECM. Since the decellularization method described here relies on diffusion of reagents into the tissue, extending this protocol to other tissues may require optimization depending upon the size and composition of the targeted tissue. The development of this method is a first step toward understanding not only the biochemical cues that exist within skeletal muscle ECM, but also the structural and mechanical signals for tissue maintenance.