RESEARCH INTERESTS: Cellular and molecular mechanisms of striated muscle physiopathology

1. PHARMACOLOGICAL, PHYSICAL, AND NUTRITIONAL INTERVENTIONS AGANIST CANCER CACHEXIA: My laboratory is focused on different approaches to counteract cancer cachexia, including pharmacological (exercise mimetics), physiological (physical activity), and nutritional (supplements) interventions in humans and animal models. 2. MYOFIBER MEMBRANE DAMAGE AND REPAIR: Duchenne Muscular Dystrophy (DMD), is a lethal genetic, muscle-wasting disease, characterized by progressive muscle fragility and weakness. The muscle membrane repair mechanism (MRM) is an active resealing pathway involving vesicle-sarcolem fusion to “patch” the compromised plasma membrane and represents a possible target to counteract muscle wasting in DMD, in which the chronic cycle of muscle degeneration-regeneration plays a pivotal role in disease progression. 3. PATENTS AND TECHNOLOGY TRASNFER: I am co-inventor of a patented procedure to produce Hsp60-enriched exosomes with exercise-mimetic activity, a product that is, therefore, called Physiactisome. Patent: Physiactisome – «Procedure for the synthesis of HSP-containing exosomes and their use against muscle atrophy and cachexia» - patent n. 102018000009235 on 8/10/2018, deposited by Università di Palermo. Owners: Università di Palermo, Università di Roma La Sapienza, Nanovector Torino, Sorbonne Université. List of inventors: Valentina Di Felice, Rosario Barone, Antonella Marino Gammazza, Campanella Claudia, Cappello Francesco, Farina Felicia, Eleonora Trovato, Daniela D’Amico, Filippo Macaluso, Dario Coletti, Sergio Adamo, Gabriele Multhoff, Paolo Gasco. International publication number WO 2020/075004 A1. This product can be exploited against muscle atrophy, since it ameliorates muscle endurance and homeostasis. The presentation of the product and the corresponding Spinoff project (iBioTHEx) was awarded the third prize at the EIT JumpStarter Grand final, Riga, Latvia, 15-17/11/2019, Health category. 4. PHYSIOPATHOLOGY OF MUSCLE TISSUES: I contribute to discovering and explaining those mechanisms underlying pathologies of the striated and smooth muscle tissues; this activity is carried out at Sorbonne University by using genetic murine models.

Cancer cachexia

Cancer cachexia
Compared to a control mouse (left) a tumor-bearing mouse (right) displays a dramatic muscle wasting. This loss of muscle mass is called cancer cachexia.

Exogenous gene expression in regenerating muscle

Exogenous gene expression in regenerating muscle
Depicted here is the over-expression of Green Fluorescent Protein (GFP, green; click on the image to access Tsien's Lab) in interstitial cells (circled), nascent myofibers (arrow) and adult fibers (arrowhead), in a regenerating Tibialis Anterior following focal injury. Laminin staining (red) highlights the basement membrane surrounding the skeletal muscle tissue, while nuclei are stained in blue. We do gene delivery by electroporation to study the regulation of muscle regeneration.

RESEARCH INTERESTS: Tissue engineering of skeletal muscle

Background and rationale.

Tissue engineering lies at the interface of regenerative medicine and developmental biology, and represent an innovative and multidisciplinary approach to build organs and tissues (Ingber and Levin, Development 2007). The skeletal muscle is a contractile tissue characterized by highly oriented bundles of giant syncytial cells (myofibers) and by mechanical resistance. Contractile, tissue-engineered skeletal muscle would be of significant benefit to patients with muscle deficits secondary to congenital anomalies, trauma, or surgery. Obvious limitations to this approach are the complexity of the musculature, composed of multiple tissues intimately intermingled and functionally interconnected, and the big dimensions of the majority of the muscles, which imply the involvement of an enormous amount of cells and rises problems of cell growth and survival (nutrition and oxygen delivery etc.). 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.” Matrix-based approaches include placing cells into compacted lattices, seeding cells onto degradable polyglycolic acid sponges, seeding cells onto acellularized whole muscles, seeding cells into hydrogels, and seeding nonbiodegradable fiber sheets. Recently, decellularized matrix from cadaveric organs has been proven to be a good scaffold for cell repopulation to generate functional hearts in mice (Ott et al. Nature Medicine 2008).

I have obtained cultures of skeletal muscle cells on conductive surfaces, which is required to develop electronic device–muscle junctions for tissue engineering and medical applications1. I aim to exploit this system for either recording or stimulation of muscle cell biological activities, by exploiting the field effect transistor and capacitor potential of the conductive substratum-cell interface. Also, we are able to create patterned dispositions of molecules and cells on gold, which is important to mimic the highly oriented pattern myofibers show in vivo.

I have found that Static magnetic fields enhance skeletal muscle differentiation in vitro by improving myoblast alignment2. Static magnetic field (SMF) interacts with mammal skeletal muscle; however, SMF effects on skeletal muscle cells are poorly investigated. 80 +/- mT SMF generated by a custom-made magnet promotes myogenic cell differentiation and hypertrophy in vitro. Finally, we have transplanted acellular scaffolds to study the in vivo response to this biomaterial3, which we want to exploit for tissue culture and regenerative medicine of skeletal muscle.

The specific aims of my current research are:

1) to increase and optimize the production and alignment of myogenic cells and myotubes in vitro;

2) to manipulate the niche of muscle stem cells aimed at ameliorating their regenerative capacity in vivo;

3) to develop muscle-electrical devices interactions. We plan to exploit the cell culture system on conductive substrates for either recording or stimulation of muscle cell biological activities, by exploiting the field effect transistor and capacitor potential of the conductive substratum-cell interface.

4) to better clarify the biological effects of Static Magnetic Fields. With the aim to characterize the molecular mechanism underlying the effects of SMF on cell differentiation and alignment we are exposing molecules and cells to SMF below 1T.
5) to produce pre-assembled, off-the-shelf skeletal muscle. We are seeding acellularized muscle scaffold with various cell types, with the goal to obtain functional muscle with vascular supply and nerves.


REFERENCES

1) Coletti D. et al., J Biomed Mat Res 2009; 91(2):370-377.

2) Coletti D. et al., Cytometry A. 2007;71(10):846-56.


3) Perniconi B. et al. Biomaterials, 2011 in press

Cultures of myotubes on a conductive surface in a parallel orientation.

Cultures of myotubes on a conductive surface in a parallel orientation.
C2C12 cells cultured on gold, by mean of adhesion to 100 nm-wide stripes coated with anti Stem Cell antigen1 (Sca1) Ab. Nuclei (blue) and actin cytoskeleton (red) staining highlights the selective cells adhesion on the Ab-coated stripes and the formation of parallel multinucleated syncytia (myotubes).

7/22/2011

ARTICLES: Perniconi et al. Biomaterials 2011

In this paper in press we show that one can transplant ghosts of tissues to obtain again the corresponding tissue in mice. Our "ghosts" are acellular scaffolds derived by whole organ decellularization (in 1% SDS) of skeletal muscles (the EDL and the TA). Therefore, they represent the extracellular matrix voided of the cellular component. We show that this biomaterial has niche properties, since it is able to support neomyogenesis once transplanted to replace the matching muscle.
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7/16/2011

LAB METHODS: transplantation of an acellular scaffold to replace the corresponding muscle



We are about to publish a paper where we characterize the in vivo response to a graft composed by an acellular scaffold obtained by a previously decellularized skeletal muscle. The grafting procedure is now available as a ppt - link embedded in the title of this post. The corresponding video on how to replace a TA with the corresponding acellular scaffold(iPod version) is available through the link in parentheses. For an alternative format, try to click here (avi version). The video is supplemented as Additional materilas to the Biomaterials article.
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LAB METHODS: Toluidine blue staining


There is no staining method as fast and informative (two for the price of one!) as the Toluidine blue staining. We use it while cryosectioning or while doing semithin sections to monitor sample quality and orientation. Toluidine specifically stains some cell and ECM features. Linked to the title of this post, you'll find our method for Toluidine staining, with references and additional examples. Fig. legend: Toluidine-stained skeletal muscle cryosections.
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Research fundings: an update...


Well...I was too pessimistic. The fundings for the Fiscal Year 2009 ("PRIN 2009") has been released by the Italian Ministry of University and Research , with a delay of only three years and not four years, as I was foreseeing.
That's good news, worth at least a bottle of Prosecco di Valdobbiadene Giustino B. by Ruggeri!
That is also a good chance to have a look at what the USA are doing. Linked to the title is the analysis of the current presidential plan for R&D in that country. President Obama requested $ 147,696 bilion for research in the current Fiscal Year. With this rate they will DOUBLE the fundings in 11 years. Linked to the title, please find the full text of the analysis of this plan.

Left:
Research & Develoment funding path in the USA

Source:
Federal Research end Development Funding - FY 2011
JF Sargent jr., coordinator, specialist in Science and Technology Policy
June 10, 2011
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THE NETWORK OF OUR COLLABORATORS 2017

THE NETWORK OF OUR COLLABORATORS 2017
We collaborate with the Myology Group and the Cochin Hospital in Paris for stem cell studies and SRF, with the Cancer Centre at Ohio State University, Columbus for studies on the mechanisms underlying cachexia, with the Neurorehabilitation Unit at University of Pisa for clinical studies, with Pharmacology and Bioinformatics at the University of Urbino for advanced statistical analyses, with the Anatomy Section at the University of Perugia and with GYN/OB at the University of Western Piedmont for studies related to circulating factors and myogenic cell responses in cachexia, with the Biotech-Med Unit at ENEA, Chemistry in Rome and Anatomy in palermo for tissue engineering applications. Functional studies are carried out in our Departement in Rome in collaboration with Musaro's laboratory.