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Skeletal Muscle Biopsy Beyond Histological Diagnosis: Ex-vivo Culturing and 3D Bioengineered model

Skeletal Muscle : Overview, Histology and Pathology

Muscle tissue represent one of the four tissue in the human body with the epithelial tissue, connective tissue and nervous tissue. Embryologically, the muscle tissue is derived by the mesoderm and it can be skeletal, cardiac or smooth tissue type. The main differences on muscle tissue types rely on the histology, function and location (Table 1)

Comparison-of-Structure-and-Properties-of-Muscle-Tissue-Types

Table 1. Comparison of Structure and Properties of Muscle Tissue Types.CC 3.0 BY OpenStax College, ‘Anatomy & Physiology’, Connexions Web site http://cnx.org/content/col11496/1.6/, Jun 19, 2013

Skeletal muscles produce voluntary movement, locomotion, maintain the posture, protect internal organs, generates heat and is responsible of facial expression. The muscle cells are also called myocyte their numbers remain constant throughout life. The cells are multinucleate and organised in muscle fibers. The striated appearance is due to the regular alternation of actin and myosin (both contractile proteins of the sarcomere, the functional units of the skeletal muscle. Each skeletal muscle fiber is innervated by a motor neuron which activate the fiber to contract through the neuromuscular junction.

Muscle diseases, disorders, and myopathies [1, 2] can be due to

-Injuries (trauma, sports or tumor excision)

-Lack of use (aging or disability)

-Fatigue

-Genetic condition: primary muscular dystrophies (Duchenne, Becker, fascioscapulohumeral muscular dystrophy, myotonic dystrophy, myotonia congenita, mitochondrial myopathies)

-Autoimmune (myasthenia gravis, polymyositis, systemic sclerosis, rheumatoid arthritis)

-Toxins (toxic myopathies)

-Inflammation (bacterial or viral myositis)

-Cancer or myopathies associates such as dermatomyositis

-Endocrine myopathies (thyrotoxicosis, hyper- or hypo- thyroidism, Cushing syndrome, vitamin D deficiency)

-Periodic paralyses

Muscle biopsy – a tool for diagnosis and muscle disease evaluation

The clinical evaluation of muscle diseases includes clinical examination, electrodiagnostic, laboratory and molecular genetic testing. The indication of muscle biopsy can be performed in the setting of quantifiable weakness and when the previous non-invasive clinical evaluation failed on identifying a certain diagnosis in neuromuscular diseases [3]. The procedure for biopsy can be open or needle biopsy, depending on the incision (5-10 mm in the needle biopsy vs >30mm in the open biopsy) [4]. There are two major categories of histopathologic abnormalities observed in muscle disease, neurogenic atrophy and myopathic changes. These structural changes often occur in tandem in myopathies [3]. The ideal amount of tissue depends on the use and the assessment technique:

-1 cm3 of frozen section for evaluate the morphology and enzyme histochemistry with light microscopy;

-5cm x 1cm of paraffin embedding tissue to evaluate the inflammation and the morphology of the inflamed cells;

-1-2 mm thick section for ultrastructural analysis with electron microscopy

-50-550 mg of tissue for biochemical testing such as for mitochondrial diseases.

Muscle biopsy beyond histology: preclinical models for pharmaceutical and toxicological studies and in regeneration medicine

Skeletal muscle tissue has a considerable capacity to regenerate thanks to the myoblasts activation, migration, proliferation, and fusion [5]. Myoblasts, progenitor of skeletal muscle, are better surviving and proliferate in whole-muscle allograft than when isolated in cell culture, also in longer term [6]. The interest of growing in vitro the skeletal muscle has been study as myoblast transfer therapy (MTT) for Duchenne muscular dystrophy exanimating the culture condition of major survival for myoblast based on effects of proteolytic enzymes, serum composition, and tissue preparation [7]. Ex vivo testing has been also suggested to study neuromuscular synapses by co-culturing spinal cord and muscle tissue, providing an adequate model system for toxic chemicals and antidotal treatment [8].

A new protocol has been described on how isolate human muscle progenitors cells (hMPCs) from skeletal muscle biopsy to address questions pertaining to skeletal muscle biology and regeneration [9]. This method has also a potential in the regenerative medicine considering the limitation of the high donor heterogeneity.

Nowadays, muscle regeneration can be also be obtained from:

-Decellularized tissue by promoting cell homing, angiogenesis, and regeneration of myofibers and motor neuron axons [10].

-Skeletal muscle tissue engineering using polymers, such as poly(ethylene glycolic) (PEG), polyglycolic acid (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(l-lactic acid) (PLLA), their copolymers (PLLA/PLGA), and polycaprolactone (PCL); natural polymers such as alginate, collagen, fibrin, or hyaluronic-acid, or a combination of the two [11-12]

3D bioprinting has a great potential in the engineering of functional 3D tissues in vitro for applications in drug discovery and regenerative medicine [13-14].

In a recent study of Gaurav Agrawal, engineered skeletal muscle tissue has been included in a skeletal muscle-on-a-chip[15]. The authors describe the architectural and structural complexities of muscle within a microfluidic device by characterising the morphology and cells and tissue formation, useful as potential alternative to animal testing in preclinical drug discovery. Further studies are requested to find adequate preclinical models for skeletal muscle diseases and treatments, also applying physiological gas and temperature condition and fluidic flow.

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References :

[1] Bradley WG, Henson RA et al, Muscle disease, Encyclopaedia Britannica, inc, September 2018, https://www.britannica.com/science/muscle-disease

[2] Agrawal, G., Aung, A., & Varghese, S. (2017). Skeletal muscle-on-a-chip: An in vitro model to evaluate tissue formation and injury. Lab on a Chip, 17(20), 3447–3461. https://doi.org/10.1039/c7lc00512a

[3] Joyce, N. C., Oskarsson, B., & Jin, L. (2015). Muscle Biopsy Evaluation in Neuromuscular Disorders Nanette. Phys Med Rehabil Clin N Am, 23(3), 609–631. https://doi.org/10.1016/j.pmr.2012.06.006.Muscle

[4] Meola G, Bugiardini E, Cardani R. Muscle biopsy. J Neurol. 2012 Apr; 259(4):601–10. [PubMed: 21805256]

[5] Lawson-Smith, M. J.; McGeachie, J. K. Experimental skeletal muscle grafts as a model of regeneration. Aust. N. Z. J. Surg. 67:35-39; 1997.

[6] Fan, Y.; Beilharz, M. W.; Grounds, M. D. A potential alternative strategy for myoblast transfer therapy: The use of sliced muscle grafts. Cell Transplant. 5:421-433; 1996

[7] Smythe, G. M., & Grounds, M. D. (2000). Exposure to tissue culture conditions can adversely affect myoblast behavior in vivo in whole muscle grafts: Implications for myoblast transfer therapy. Cell Transplantation, 9(3), 379–393. https://doi.org/10.1177/096368970000900309

[8] Drexler, B., Seeger, T., Grasshoff, C., Thiermann, H., & Antkowiak, B. (2011). Long-term evaluation of organophosphate toxicity and antidotal therapy in co-cultures of spinal cord and muscle tissue. Toxicology Letters, 206(1), 89–93. https://doi.org/10.1016/j.toxlet.2011.04.010

[9] Gheller, B. J., Blum, J., Soueid-Baumgarten, S., Bender, E., Cosgrove, B. D., & Thalacker-Mercer, A. (2019). Isolation, culture, characterization, and differentiation of human muscle progenitor cells from the skeletal muscle biopsy procedure. Journal of Visualized Experiments,2019(150), 1–10. https://doi.org/10.3791/59580

[10] Urciuolo, A., & De Coppi, P. (2018). Decellularized tissue for muscle regeneration. International Journal of Molecular Sciences, 19(8), 1–11. https://doi.org/10.3390/ijms19082392

[11] Saxena, A.K.; Marler, J.; Benvenuto, M.; Willital, G.H.; Vacanti, J.P. Skeletal muscle tissue engineering using isolated myoblasts on synthetic biodegradable polymers: Preliminary studies. Tissue Eng. 1999, 5, 525–532. [CrossRef] [PubMed]

[12] Page, R.L.; Malcuit, C.; Vilner, L.; Vojtic, I.; Shaw, S.; Hedblom, E.; Hu, J.; Pins, G.D.; Rolle, M.W.; Dominko, T. Restoration of Skeletal Muscle Defects with Adult Human Cells Delivered on Fibrin Microthreads. Tissue Eng. Part A 2011, 17, 2629–2640. [CrossRef] [PubMed]

[13] Laternser, S., Keller, H., Leupin, O., Rausch, M., Graf-Hausner, U., & Rimann, M. (2018). A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues. SLAS Technology, 23(6), 599–613. https://doi.org/10.1177/2472630318776594

[14] Carosio, S., Barberi, L., Rizzuto, E., Nicoletti, C., Prete, Z. Del, & Musarò, A. (2013). Generation of eX vivo-vascularized Muscle Engineered Tissue (X-MET). Scientific Reports, 3, 1–9. https://doi.org/10.1038/srep01420

[15] Agrawal, G., Aung, A., & Varghese, S. (2017). Skeletal muscle-on-a-chip: An in vitro model to evaluate tissue formation and injury. Lab on a Chip, 17(20), 3447–3461. https://doi.org/10.1039/c7lc00512a

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