Barbara Perniconi

Native extracellular matrix: A new scaffolding platform for repair of damaged muscle

Effective clinical treatments for volumetric muscle loss resulting from traumatic injury or resection of a large amount of muscle mass are not available to date. Tissue engineering may represent an alternative treatment approach. Decellularization of tissues and whole organs is a recently introduced platform technology for creating scaffolding materials for tissue engineering and regenerative medicine. The muscle stem cell niche is composed of a three-dimensional architecture of fibrous proteins, proteoglycans, and glycosaminoglycans, synthesized by the resident cells that form an intricate extracellular matrix (ECM) network in equilibrium with the surrounding cells and growth factors. A consistent body of evidence indicates that ECM proteins regulate stem cell differentiation and renewal and are highly relevant to tissue engineering applications. The ECM also provides a supportive medium for blood or lymphatic vessels and for nerves. Thus, the ECM is the natures ideal biological scaffold material. ECM-based bioscaffolds can be recellularized to create potentially functional constructs as a regenerative medicine strategy for organ replacement or tissue repopulation. This article reviews current strategies for the repair of damaged muscle using bioscaffolds obtained from animal ECM by decellularization of small intestinal submucosa (SIS), urinary bladder mucosa (UB), and skeletal muscle, and proposes some innovative approaches for the application of such strategies in the clinical setting.

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 environment in 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.

Muscle Extracellular Matrix Scaffold Is a Multipotent Environment

The multipotency of scaffolds is a new concept. Skeletal muscle acellular scaffolds (MAS) implanted at the interface of Tibialis Anterior/tibial bone and masseter muscle/mandible bone in a murine model were colonized by muscle cells near the host muscle and by bone-cartilaginous tissues near the host bone, thus highlighting the importance of the environment in directing cell homing and differentiation. These results unveil the multipotency of MAS and point to the potential of this new technique as a valuable tool in musculo-skeletal tissue regeneration.

Skeletal muscle tissue engineering: best bet or black beast?

Skeletal muscle possesses a remarkable self-repair capacity whose underlying mechanisms have been thoroughly investigated (Vandenburgh et al., 1988; De Arcangelis et al., 2003; Musaro et al., 2007; Moresi et al., 2008, 2009) with a view to stimulating in situ regeneration. It is, however, unable to restore volumetric tissue loss as a consequence of trauma, congenital defects, ablation, or denervation. This is the rationale behind the creation of new skeletal muscle through tissue engineering (TE). While most review articles announce that skeletal muscle TE is advancing and is readily translatable, it seems clear that engineered skeletal muscle is still lagging far behind other tissues if placed within a clinical practice context. Why is it not yet possible to transplant off-the-shelf, functional muscles into patients? Major difficulties encountered in skeletal muscle TE on the whole organ scale are currently being addressed. These include the following: the large size of human organs has been overcome by studies on rabbit, dogs and humans (Badylak et al., 1998; Rossi et al., 2010; Badylak et al., 2013); numerous myogenic cell populations of muscle and non-muscle origin (Rossi et al., 2010) are now available as cell sources; scaffolds with a specific 3D orientation are obtained through organ decellularization, freeze drying and electrospinning of synthetic or natural materials (Klumpp et al., 2010); function has been proven for several of these engineered muscle constructs (Mudera et al., 2010; Carosio et al., 2013). Even some of the interactions between muscle and other organs have been addressed: the muscular-tendinous junction can be restored by suturing the residual tendon proximal extremity of muscle-derived acellular scaffolds to the host tendon (Perniconi et al., 2011); the vascular bed can be reconstructed in striated muscles (Koffler et al., 2011; Carosio et al., 2013), particularly if the elegant approach proposed by Ott et al. for decellularized heart is applied (Ott et al., 2008). Muscles, however, remain very challenging organs to rebuild. Muscle hierarchic architecture and heterogeneous cell composition have not yet been sufficiently investigated by either in vitro or in vivo studies. Moreover, innervation by the somato-motor system has not been addressed at all. We believe that the latter issue will be the hardest to address; since we are still unable to induce normal re-innervation following motor neuron injury, the successful innervation of a new, pre-built organ transplanted in vivo is unlikely to be straightforward. Lastly, even some basic aspects of muscle TE, such as graft bioactivity and integration, which are often claimed to be established, are actually still poorly understood; for instance, the novel idea of cryptic peptides released by the extra-cellular matrix (ECM) while it is being biodegraded and remodeled (Grayson et al., 2009) opens new avenues for the exploration of ECM component bioactivity. This need to gain a better knowledge of the properties of the ECM and bioactive molecules used in TE applications is indeed the rationale of dedicated publications, such as this Frontiers in Muscle Physiology Special Issue. While obtaining a fully functional, innervated human skeletal muscle from TE that may be used for clinical purposes remains a long-term goal, here we propose two applications for engineered muscle that are no less ambitious and that may be achieved and exploited in the short term. As stated by Grayson et al., the biomimetic effort being made within the context of skeletal muscle TE is mostly aimed at: (a) the creation of functional tissue grafts for regenerative medicine applications in vivo; (b) the generation of experimental models in vitro for studies on stem cells, development, and disease (where engineered tissues can serve as advanced 3D models). As far as organ replacement in vivo is concerned, we expect very specific TE-based interventions, such as the application of muscle flaps and the generation of minute individual muscles, to be immediately successful, whereas the mass production of large muscles involved in chronic and global muscle wasting diseases is less unlikely to be so. One example of organ replacement that is likely to be successful is the Stapedius, which is the smallest and weakest skeletal muscle in the human body. It stabilizes the stapes, a very small bone in the inner hear, and is innervated by the facial nerve. Dysfunctions in the Stapedius induce hyperacusis or other sound perception defects that are clinically relevant. While the problem of innervation is likely to persist, the engineering and grafting of this small and simple muscle to replace the diseased one (often due to a local defect) appears to be highly feasible. As far as in vitro studies are concerned, it is self-evident that bidimensional cultures are very limited insofar as the physiological 3D tissue organization they yield is somewhat approximate. Muscle TE was initially designed for in vitro studies, when Vandenburgh et al. introduced the 3D cultivation of primary myoblasts in collagen gel and generated contracting muscle tissue in vitro for the first time in 1988 (Vandenburgh et al., 1988). A progressive increase in the architectural complexity of ECM and cells in tissue-culture grade constructs is likely to provide adequate experimental models for the in vitro study of phenomena that are specific to the in vivo situation [e.g., stem cell niche, tissue regeneration, aging (Sharples et al., 2012)]. With regard to the two applications described above, it should be borne in mind that the goals and approaches involved in building engineered muscle tissue may not be the same owing to the significant differences between in vitro and in vivo TE strategies (reviewed by Rossi et al., 2010). For all these reasons, we believe that the best bet for skeletal muscle TE is to focus on specific, anatomically defined solutions or on 3D in vitro modeling of muscle tissue for basic and applied research. We are confident that we will eventually be able to transform the black beast (i.e., striated muscle tissue engineering) into the best bet (i.e., a successful clinical practice based on engineered muscles). However, for more ambitious muscle TE applications, there may still be a long way to go.

Long-term exposure to Myozyme results in a decrease of anti-drug antibodies in late-onset Pompe disease patients

Immunogenicity of recombinant human acid-alpha glucosidase (rhGAA) in enzyme replacement therapy (ERT) is a safety and efficacy concern in the management of late-onset Pompe disease (LOPD). However, long-term effects of ERT on humoral and cellular responses to rhGAA are still poorly understood. To better understand the impact of immunogenicity of rhGAA on the efficacy of ERT, clinical data and blood samples from LOPD patients undergoing ERT for >4 years (n = 28) or untreated (n = 10) were collected and analyzed. In treated LOPD patients, anti-rhGAA antibodies peaked within the first 1000 days of ERT, while long-term exposure to rhGAA resulted in clearance of antibodies with residual production of non-neutralizing IgG. Analysis of  T cell responses to rhGAA showed detectable T cell reactivity only after in vitro restimulation. Upregulation of several cytokines and chemokines was detectable in both treated and untreated LOPD subjects, while IL2 secretion was detectable only in subjects who received ERT. These results indicate that long-term ERT in LOPD patients results in a decrease in antibody titers and residual production of non-inhibitory IgGs. Immune responses to GAA following long-term ERT do not seem to affect efficacy of ERT and are consistent with an immunomodulatory effect possibly mediated by regulatory T cells.

Static magnetic fields modulate X-ray-induced DNA damage in human glioblastoma primary cells.

Although static magnetic fields (SMFs) are used extensively in the occupational and medical fields, few comprehensive studies have investigated their possible genotoxic effect and the findings are controversial. With the advent of magnetic resonance imaging-guided radiation therapy, the potential effects of SMFs on ionizing radiation (IR) have become increasingly important. In this study we focused on the genotoxic effect of 80 mT SMFs, both alone and in combination with (i.e. preceding or following) X-ray (XR) irradiation, on primary glioblastoma cells in culture. The cells were exposed to: (i) SMFs alone; (ii) XRs alone; (iii) XR, with SMFs applied during recovery; (iv) SMFs both before and after XR irradiation. XR-induced DNA damage was analyzed by Single Cell Gel Electrophoresis assay (comet assay) using statistical tools designed to assess the tail DNA (TD) and tail length (TL) as indicators of DNA fragmentation. Mitochondrial membrane potential, known to be affected by IR, was assessed using the JC-1 mitochondrial probe. Our results showed that exposure of cells to 5 Gy of XR irradiation alone led to extensive DNA damage, which was significantly reduced by post-irradiation exposure to SMFs. The XR-induced loss of mitochondrial membrane potential was to a large extent averted by exposure to SMFs. These data suggest that SMFs modulate DNA damage and/or damage repair, possibly through a mechanism that affects mitochondria.

Multidisciplinary care allowing uneventful vaginal delivery in a woman with Pompe disease.

Pregnancy and delivery are challenging in women affected by Pompe disease with respiratory involvement. We describe a 28-year-old woman, who continued to receive enzyme replacement therapy during pregnancy and had an uneventful vaginal birth. Before pregnancy the patients vital capacity was 52% in sitting position and 51% in supine position. At 32 weeks gestation her vital capacity in sitting position was 46% and 35% in supine position. Nocturnal non-invasive mechanical ventilation was introduced at this time. Labor was induced at 34 weeks following premature rupture of membranes, under epidural anesthesia. A 2590 g healthy baby was delivered by vacuum extraction. Assisted ventilation was continued throughout labor and post-partum. This observation suggests a successful pregnancy and a normal vaginal delivery can be achieved in patients with symptomatic Pompe Disease, provided multidisciplinary care is offered.

The impact of enzyme replacement therapy on the progression of Pompe disease

G.P.8 An international, phase 3, switchover study of reveglucosidase alfa (BMN 701) in subjects with late-onset Pompe disease B. Schoser *, B. Byrne , F. Eyskens , T. Hiwot , D. Hughes , J. Kissel , E. Mengel , T. Mozaffar , A. Pestronk , M. Roberts , K. Sivakumar , J. Statland , P. Young , C. Heusner , W. Dummer 14 1 Ludwig Maximilians University, Munich, Germany; 2 University of Florida School of Medicine, Gainesville, FL, USA; 3 University Hospital of Antwerp, Antwerp, Belgium; 4 University Hospital Birmingham, Birmingham, UK; 5 Royal Free & University College Medical School, London, UK; 6 Ohio State University, Columbus, OH, USA; 7 Johannes Gutenberg University Mainz, Mainz, Germany; 8 University of California, Irvine, CA, USA; 9 Washington University School of Medicine, Saint Louis, MO, USA; 10 Salford Royal NHS Foundation Trust, Salford, UK; 11 Neuromuscular Research Center, Scottsdale, AZ, USA; 12 University of Kansas Medical Center, Kansas City, KS, USA; 13 University Hospital Munster, Munster, Germany; 14 BioMarin Pharmaceutical Inc., Novato, CA, USA

Vaginal birth in a patient with Pompe disease

G.P.1 Cytokine and chemokine profiling shows rapid activation of the immune system following enzyme replacement therapy in Pompe disease E. Masat *, P. Laforet , D. Amelin , P. Veron , B. Perniconi , N. Taouagh , O. Benveniste , F. Mingozzi 1 1 University Pierre and Marie Curie, INSERM, UMR974 Paris, France; 2 AP-HP, Pitie-Salpetriere University Hospital, Paris-Est Neuromuscular Center, Paris, France; 3 Genethon, Evry, France; 4 AP-HP, Pitie-Salpetriere University Hospital, Institute of Myology, Paris, France; 5 AP-HP, Pitie-Salpetriere University Hospital, Department of Internal Medicine and Clinical Immunology, Paris, France