Biologic scaffold composed of skeletal muscle extracellular matrix
Introduction
Biologic scaffold materials composed of extracellular matrix (ECM) are typically produced by decellularization of mammalian tissues such as urinary bladder, dermis, or small intestine [1] and have been shown to facilitate the functional reconstruction of several tissue types [2], [3] including the lower urinary tract [4], [5], heart and vascular structures [6], [7], esophagus [8], [9], and musculoskeletal tissues [10], [11], [12], [13], among others. The mechanisms by which constructive remodeling occurs include the recruitment of multipotential stem and progenitor cells to the site of scaffold placement [14], [15], promotion of a favorable M2 macrophage phenotype at the host tissue/bioscaffold interface [16], regional angiogenesis [17], and mitogenesis [15], [18]. These tissue derived biologic scaffolds are frequently used in non-homologous anatomic sites, but recent studies have suggested that biologic scaffolds derived from site specific homologous tissues such as liver and lung may be better suited for constructive tissue remodeling than non-site specific tissue sources [19], [20], [21], [22], [23], [24], [25].
Muscle tissues, including cardiac, skeletal, and smooth muscle, respond favorably when biologic scaffolds are used for their reconstruction following injury [11], [12]. To date, there have been several attempts to isolate and process skeletal muscle ECM (M-ECM) [26], [27], [28], [29], [30], [31], [32], [33]. Most of these attempts have involved the decellularization of intact rodent muscles or the extraction of rodent muscle ECM proteins, with varying degrees of success. DeQuach et al. [33] did show that proteins extracted from a decellularized porcine muscle matrix retain bioactivity. None of these studies have provided a detailed characterization of the intact M-ECM scaffold derived from a large animal tissue source, nor have any of these studies applied stringent decellularization criteria in the development of the decellularization process. The objectives of the present study were to (1) determine a method for decellularization of skeletal muscle and characterize the structure and composition of the resulting ECM, and (2) to compare the in-vitro bioactivity and in-vivo remodeling properties of skeletal muscle ECM vs. non-muscle ECM, specifically SIS, in a rodent model of abdominal wall muscle repair.
Section snippets
Overview of study design
Canine skeletal muscle was harvested and decellularized by enzymatic and chemical methods. The resulting M-ECM was then assessed for biochemical and structural composition, the cell response in vitro, and the in vivo remodeling characteristics in a rat abdominal wall defect model. ECM composed of porcine small intestinal submucosa (SIS) was used for comparison purposes. All animal experiments were conducted in accordance to University of Pittsburgh Institutional Animal Care and Use Committee
Verification of decellularization
The amount and size of residual DNA content after decellularization for each preparation of M-ECM prepared was quantified and is presented in Fig. 1. Histologic analysis of M-ECM (Fig. 1A and C) showed no evidence of intact nuclear material on H&E or DAPI as compared to native muscle (Fig. 1B and D). After decellularization, there was 7.42 + 1.67 ng DNA/mg dry weight compared to the 1549 ± 489 ng DNA/mg dry weight found in native muscle tissue (Fig. 1E). There were no clearly visible bands of
Discussion
A method for the preparation of skeletal muscle ECM scaffolds from a large animal tissue source is described in the present study. The M-ECM scaffold was shown to be thoroughly decellularized by established criteria while simultaneously preserving many of the components found in the native ECM. The bioactivity of the scaffold was evaluated and shown to affect the proliferative potential of muscle progenitor cells in vitro as well as the induction of a constructive remodeling response in vivo.
Conclusions
A M-ECM scaffold can be prepared from a large animal source using an enzymatic and chemical processing method. The M-ECM conforms with established decellularization criteria while preserving factors found in native muscle ECM that may be beneficial to the host remodeling response. The M-ECM exerts biologic effects on myogenic cells in vitro and promotes positive remodeling characteristics in a rodent muscle defect model. However, when compared to the nonhomologous SIS there was no detectable
Acknowledgments
Funding for this work was provided by the Advanced Regenerative Medicine (ARM III) grant W81XWH-07-1-0415. Matthew Wolf was partially supported by the NIH-NHLBI training grant (T32-HL76124-6) entitled “Cardiovascular Bioengineering Training Program” through the University of Pittsburgh Department of Bioengineering. The authors would like to thank Deanna Rhoads and the McGowan Histology Center for histologic section preparation, the Center for Biologic Imaging at the University of Pittsburgh for
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