Muscle acellular scaffold as a biomaterial: Effects on C2C12 cell differentiation and interaction with the murine host environment

The extracellular matrix (ECM) of decellularized organs possesses the characteristics of the ideal tissue-engineering scaffold (i.e., histocompatibility, porosity, degradability, non-toxicity). We previously observed that the muscle acellular scaffold (MAS) is a pro-myogenic environmentin vivo. In order to determine whether MAS, which is basically muscle ECM, behaves as a myogenic environment, regardless of its location, we analyzed MAS interaction with both muscle and non-muscle cells and tissues, to assess the effects of MAS on cell differentiation. Bone morphogenetic protein treatment of C2C12 cells cultured within MAS induced osteogenic differentiation in vitro, thus suggesting that MAS does not irreversibly commit cells to myogenesis.In vivo MAS supported formation of nascent muscle fibers when replacing a muscle (orthotopic position). However, heterotopically grafted MAS did not give rise to muscle fibers when transplanted within the renal capsule. Also, no muscle formation was observed when MAS was transplanted under the xiphoid process, in spite of the abundant presence of cells migrating along the laminin-based MAS structure. Taken together, our results suggest that MAS itself is not sufficient to induce myogenic differentiation. It is likely that the pro-myogenic environment of MAS is not strictly related to the intrinsic properties of the muscle scaffold (e.g., specific muscle ECM proteins). Indeed, it is more likely that myogenic stem cells colonizing MAS recognize a muscle environment that ultimately allows terminal myogenic differentiation. In conclusion, MAS may represent a suitable environment for muscle and non-muscle 3D constructs characterized by a highly organized structure whose relative stability promotes integration with the surrounding tissues. Our work highlights the plasticity of MAS, suggesting that it may be possible to consider MAS for a wider range of tissue engineering applications than the mere replacement of volumetric muscle loss

Decellularised skeletal muscles allow functional muscle regeneration by promoting host cell migration

Pathological conditions affecting skeletal muscle function may lead to irreversible volumetric muscle loss (VML). Therapeutic approaches involving acellular matrices represent an emerging and promising strategy to promote regeneration of skeletal muscle following injury. Here we investigated the ability of three different decellularised skeletal muscle scaffolds to support muscle regeneration in a xenogeneic immune-competent model of VML, in which the EDL muscle was surgically resected. All implanted acellular matrices, used to replace the resected muscles, were able to generate functional artificial muscles by promoting host myogenic cell migration and differentiation, as well as nervous fibres, vascular networks, and satellite cell (SC) homing. However, acellular tissue mainly composed of extracellular matrix (ECM) allowed better myofibre three-dimensional (3D) organization and the restoration of SC pool, when compared to scaffolds which also preserved muscular cytoskeletal structures. Finally, we showed that fibroblasts are indispensable to promote efficient migration and myogenesis by muscle stem cells across the scaffolds in vitro. This data strongly support the use of xenogeneic acellular muscles as device to treat VML conditions in absence of donor cell implementation, as well as in vitro model for studying cell interplay during myogenesis

Molecular, cellular and physiological characterization of the cancer cachexia-inducing C26 colon carcinoma in mouse

BACKGROUND: The majority of cancer patients experience dramatic weight loss, due to cachexia and consisting of skeletal muscle and fat tissue wasting. Cachexia is a negative prognostic factor, interferes with therapy and worsens the patients' quality of life by affecting muscle function. Mice bearing ectopically-implanted C26 colon carcinoma are widely used as an experimental model of cancer cachexia. As part of the search for novel clinical and basic research applications for this experimental model, we characterized novel cellular and molecular features of C26-bearing mice. METHODS: A fragment of C26 tumor was subcutaneously grafted in isogenic BALB/c mice. The mass growth and proliferation rate of the tumor were analyzed. Histological and cytofluorometric analyses were used to assess cell death, ploidy and differentiation of the tumor cells. The main features of skeletal muscle atrophy, which were highlighted by immunohistochemical and electron microscopy analyses, correlated with biochemical alterations. Muscle force and resistance to fatigue were measured and analyzed as major functional deficits of the cachectic musculature. RESULTS: We found that the C26 tumor, ectopically implanted in mice, is an undifferentiated carcinoma, which should be referred to as such and not as adenocarcinoma, a common misconception. The C26 tumor displays aneuploidy and histological features typical of transformed cells, incorporates BrdU and induces severe weight loss in the host, which is largely caused by muscle wasting. The latter appears to be due to proteasome-mediated protein degradation, which disrupts the sarcomeric structure and muscle fiber-extracellular matrix interactions. A pivotal functional deficit of cachectic muscle consists in increased fatigability, while the reported loss of tetanic force is not statistically significant following normalization for decreased muscle fiber size. CONCLUSIONS: We conclude, on the basis of the definition of cachexia, that ectopically-implanted C26 carcinoma represents a well standardized experimental model for research on cancer cachexia. We wish to point out that scientists using the C26 model to study cancer and those using the same model to study cachexia may be unaware of each other's works because they use different keywords; we present strategies to eliminate this gap and discuss the benefits of such an exchange of knowledge

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

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 hoste-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 þ4 C or þ37 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

TISSUE-ENGINEERED SKELETAL MUSCLE: PRELIMINARY STUDIES FOR IN VIVO TRANSPLANTATION

Contractile, tissue-engineered skeletal muscle would be a significant benefit to patients with muscle deficits secondary to congenital anomalies, trauma, or surgery. For the reconstructive interventions, tissue-engineered skeletal muscle may offer reduced donor-site morbidity and unlimited supply of tissue. Obvious limitations to this approach are the complexity of the musculature, composed of multiple tissues intimately intermingled and functionally interconnected. Two major approaches are followed to address these issues. Self-assembled skeletal muscle constructs are produced in vitro by delaminating sheets of cocultured myoblasts and fibroblasts, which results in contractile cylindrical “myooids.” Alternatively, matrix-based approaches include placing cells into scaffold of various origin, including organic polymers of synthetic and of biological origin. Our main goal is to generate a functional, implantable skeletal muscle containing different tissues, pre-assembled in vitro on a scaffold derived from a cadaveric muscle

Decellularized scaffolds from skeletal muscle are a suitable environment for myogenesis in vivo

Background. Skeletal muscle defects arising from traumatic or pathological conditions may require surgical interventions. In our previous studies we described the interactions between a decellularized scaffold derived from a murine skeletal muscle and an isogenic mouse host. Our research is focused on the interaction of the grafted scaffold with other tissues, i.e immune and nervous systems. In addition, we aim to build off-the-shelf, transplantable constructs by culturing myogenic cells into the scaffold before transplantation. Methods. We decellularized Tibialis Anterior muscle of cadaveric origin. The decellularization process was achieved by incubation of dissected muscles in a detergent solution. To replace homologous muscles, we orthotopically transplanted decellularized scaffolds, by suturing them to the host tendon extremities following TA removal. With this approach the constructs were analyzed in regard to histocompatibility, bioactivity, degradability, toxicity in vivo at different times from transplantation. Alternatively, before transplantation the scaffolds were pre-seeded with myogenic cells in vitro. Results and conclusions. The procedure to produce acellular scaffolds from cadaveric skeletal muscles preserves the extracellular matrix and its anatomical pattern. The transplanted acellular scaffold is readily colonized by both inflammatory and myogenic stem cells, as demonstrated by the expression of stem cell markers, followed by the formation of muscle fibers. The latter show centrally located nuclei and express muscle-specific myosin. Cytofluorimetric analysis of the inflammatory cells population shows that CD45+ cells are mostly represented by macrophages, even though T lymphocytes and granulocytes are also present. So likely macrophages play a major role in acellular muscle graft integration and remodeling. Functionality of skeletal muscle is strictly dependent on myofibers spatial orientation and on innervation. Preliminary data suggest the presence of neuro-muscular junctions in the proximity of the cells that populate the grafted scaffold, suggesting the potential for re-innervation of the implant. The integration of the scaffold can likely be boosted by previous myogenic cell colonization, so we delivered satellite cells to the acellular scaffolds and noted the formation of muscle fibers into the construct in culture. Our studies show that scaffolds derived from decellularized muscles are a highly myogenic environment and may represent an innovative tool for skeletal muscle regenerative medicine