Elsevier

Biomaterials

Volume 32, Issue 31, November 2011, Pages 7870-7882
Biomaterials

The pro-myogenic environment provided by whole organ scale acellular scaffolds from skeletal muscle

https://doi.org/10.1016/j.biomaterials.2011.07.016Get rights and content

Abstract

In the pursuit of a transplantable construct for the replacement of large skeletal muscle defects arising from traumatic or pathological conditions, several attempts have been made to obtain a highly oriented, vascularized and functional skeletal muscle. Acellular scaffolds derived from organ decellularization are promising, widely used biomaterials for tissue engineering. However, the acellular skeletal muscle extra cellular matrix (ECM) has been poorly characterized in terms of production, storage and host–donor interactions. We have produced acellular scaffolds at the whole organ scale from various skeletal muscles explanted from mice. The acellular scaffolds conserve chemical and architectural features of the tissue of origin, including the vascular bed. Scaffolds can be sterilely stored for weeks at +4 °C or +37 °C in tissue culture grade conditions. When transplanted in wt mice, the grafts are stable for several weeks, whilst being colonized by inflammatory and stem cells. We demonstrate that the acellular scaffold per se represents a pro-myogenic environment supporting de novo formation of muscle fibers, likely derived from host cells with myogenic potential. Myogenesis within the implant is enhanced by immunosuppressive treatment. Our work highlights the fundamental role of this niche in tissue engineering application and unveils the clinical potential of allografts based on decellularized tissue for regenerative medicine.

Introduction

A hallmark of biomaterials, an evolving concept recently reviewed by Williams [1], is the in vivo interaction with the host’s biological components. The various strategies used to obtain biomaterials for tissue engineering (TE) applications include allografts produced by the recellularization of previously decellularized tissues. Such allografts are widely used, especially for in vivo replacement of tissues and organs whose spatial organization and biochemical composition are complex. The decellularization of an explanted tissue can be achieved through various approaches, all of which eliminate the cellular compartment and leave a spatially and chemically preserved ECM [2]. These approaches have been successfully used to produce transplantable vessels, skin and cardiac tissues [3], [4], [5]. The ECM is, by definition, nature’s ideal biological scaffold material. Indeed, it is specifically synthesized by the resident cells of each tissue and is obviously biocompatible since host cells produce their own matrix. The ECM also provides a supportive medium for blood or lymphatic vessels and for nerves. As reviewed in detail by Badylak, the ECM possesses all of the characteristics of the ideal tissue-engineered scaffold or biomaterial [6]. However, since complex three-dimensional organization of the structural and functional molecules that make up the ECM has not yet been fully characterized, synthesis of this material cannot be fully reproduced in the laboratory. ECM can be obtained from allogenic or syngenic donors, which may pose the histocompatibility and resorption problems that are typical of allografts. ECM scaffold materials that are resistant to degradation appear to elicit a pro-inflammatory macrophage (M1)-like response, whereas the anti-inflammatory (M2) macrophage phenotype prevails in native ECM scaffold materials, which are consequently readily degraded [7].

Recent advances in skeletal muscle TE have opened new perspectives for the replacement of this tissue in common clinical applications, such as traumatic injury, extended tissue ablation or denervation [8]. Key issues in skeletal muscle TE are the composition and architecture of the ECM of this tissue, which is characterized by a highly ordered and hierarchical organization of muscle fibers. Several attempts have been made to address this issue by seeding fibrillar matrices with myoblasts or myogenic stem cells [8]. Skeletal muscle constructs have been obtained by using the ECM deriving from decellularized tissue. In particular, acellular muscles have been used by Borschel et al. as a substratum for C2C12 myoblast cultures, thus producing constructs capable of longitudinal contractile force upon electrical stimulation [9]. The same authors have obtained vascularized constructs by culturing C2C12 cells in a fibrinogen hydrogel contained within cylindrical silicone chambers and transplanting them around the femoral vessels in isogeneic adult recipient rats [9]. Patches of homologous muscle acellular matrix seeded with autologous myoblasts have been used to repair abdominal wall defects in rodents [10]. Minced muscle replaced in its bed has been shown to effectively regenerate fibers, though such fibers are spatially disorganized probably owing to the loss of ECM spatial orientation [11]. ECM and growth factors deeply affect various aspects of cell behavior, including survival, proliferation and differentiation, and are therefore key issues in TE applications [12]. In both pathological conditions, such as Duchenne’s muscular dystrophy, and healthy conditions, such as after strenuous exercise, skeletal muscle tissue is maintained and repaired through regeneration [13]. As regeneration is strongly influenced by ECM and growth factors, ECM extracts have been used to coat culture dishes to induce muscle differentiation in vitro [14]. The role of growth factors in muscle regeneration has also been investigated in depth both in vitro and in vivo [15], [16], [17]. In order to promote host–donor tissue integration and vascularization, engineered factor VIII-releasing synthetic fibers have been used as a scaffold for myoblast cell culture before transplantation of the constructs into murine recipients [18].

While the generation of a whole functional, bioengineered rodent heart has demonstrated that it is possible to produce highly complex whole organs in vitro [5], a functional, anatomically defined skeletal muscle transplantable in vivo has not yet been produced. Few studies have evaluated and characterized the host immune response to non-autologous ECM scaffold materials. To this purpose, we generated an acellular scaffold from skeletal muscle and transplanted it into syngeneic hosts. This approach allowed us to extensively characterize histocompatibility, bioactivity and integration of acellular scaffolds in a murine model.

Section snippets

Animals

Adult sex-matched BALB/C mice were used throughout this study as both donors and hosts. For specific experiments, adult nude athymic mice (strain NU/NU Crl:NU-Foxn1nu, Charles River, Milano, Italy) were used. Mice were treated according to the guidelines of the Institutional Animal Care and Use Committee. Donor animals were sacrificed before skeletal muscle removal, while host animals were anesthetized before muscle dissection and replacement with the acellular scaffold. The transplantation

Acellular scaffold generation from skeletal muscle

A protocol based on a sequence of incubations in iso-osmotic and then hyposmotic detergent solutions was proposed some years ago to make acellular EDL muscle scaffolds from adult mice [9]. More recently, it has been shown that incubation in 1% SDS in deionized water completely decellularizes a whole murine heart [5]. We applied the latter method to two murine skeletal muscles, the EDL and the TA, which differ above all on account of the disparity in the size of their masses, which are 10 and 40 

Discussion

An increasing amount of interest is being expressed in tissue engineering and regenerative medicine as a means of restoring or replacing lost or malfunctioning tissues and organs through the use of cells and biomaterial scaffolds [27]. Adopting a widely used approach, either autologous or heterologous cells are cultured in a biocompatible three-dimensional porous scaffold supplemented with growth factors to regenerate new tissues or organs. The scaffold provides necessary interim support for

Conclusion

Allografts of acellular scaffold derived from a whole skeletal muscle explanted from mice and orthotopically transplanted in wt mice remain stable for several weeks, whilst being colonized by inflammatory and stem cells. Acellular scaffolds per se represent a pro-myogenic environment supporting de novo formation of muscle fibers, likely derived from host cells with myogenic potential. Inflammation by immunosuppressive procedures increases the volume of skeletal muscle fibers, thereby improving

Acknowledgments

We gratefully acknowledge the technical help provided by Carla Ramina and Fabrizio Padula (Sapienza University of Rome) for the confocal and flow cytometry analysis, respectively. We thank Giovanna Marazzi and David Sassoon, who have provided the 370 (anti-PW1) Ab. The authors are also grateful to Dario Rossi for his multimedia advice and expertise. Grant support from the following institution is acknowledged: AFM, Italian MIUR, Sapienza University of Rome to SA; UPMC Emergence to DC.

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    1

    Contributed equally to this work.

    2

    2010 Student Award of the International Society for the Advancement of Cytometry.

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