We explored the effect of poly(L-lactic acid) (PLLA) containing various percentages (0.1, 0.5, 1, and 3?wt.%) of multi walled carbon nanotubes (MWCNTs) on the myogenic differentiation of C2C12 murine myoblast progenitor cells. We showed that all PLLA/MWCNTs nanocomposite materials support the myotubes formation more efficiently than neat PLLA as indicated by the high expression of the most significant myogenic markers: MyoD, Myosin Heavy Chain, dimension of myofibres, and fusion myogenic index. Interestingly, we note that both MyoD and myogenic fusion index levels were in the order 0.1?MWCNTs = 0.5?MWCNTs > 1?MWCNTs > 3?MWCNTs > neat PLLA, suggesting that the amount of MWCNTs influenced the cell differentiation. 1. Introduction The goal of regenerative medicine is the generation of a biohybrid organ culture that closely mimics the physiological tissue developmental conditions through the combination of smart biomaterials and stem/precursor cells that could re-establish the metabolic alteration [1, 2]. It is hypothesised that stem/precursor cells could be able to convert the mechanical cues of biomaterials on biochemical signals and in turn modulate their fate. This phenomenon called mechanotransduction is under investigation intensively [2–4]. In this regard we have evaluated the interaction of stem cells and biomaterials showing that stem cells can act as mechanosensitive units responding to the material characteristic specifically. Thus adult, embryonic and induced pluripotent stem cells respond to nanocomposite poly(L-lactic acid) (PLLA) fibrous mats containing 1?wt.% to 8?wt.% amounts of calcium deficient hydroxyapatite with osteogenic differentiation [5], whereas human bone marrow mesenchymal stem cells (hBM-MSCs) respond to hydrogenated amorphous carbon with groove surface nanotopography with acquisition of neuronal characteristics [6]. Recently in our laboratory we have successfully developed and characterized the conductive nanocomposite films based on PLLA containing various percents (0.1, 0.5, 1, 3?wt.%) of multi walled carbon nanotubes (MWCNTs) (namely PLLA/MWCNTs: 0.1?MWCNTs, 0.5?MWCNTs, 1?MWCNTs, and 3?MWCNTs) [7]. PLLA is a biocompatible polymer matrix in nanocomposite production characterized by peculiar mechanical properties, adjustable degradation rate, and ease of manufacturing [8, 9]. Carbon nanotubes are considered perfect reinforcing agents for high-strength and conductive polymer composites, because of their mechanical response and high electrical conductivity [10]. Thus PLLA/MWCNTs nanocomposites provide the possibility to
References
[1]
S. J. Lee and A. Atala, “Scaffold technologies for controlling cell behavior in tissue engineering,” Biomedical Materials, vol. 8, no. 1, Article ID 010201, 2013.
[2]
B. Inan? and Y. M. El?in, “Stem cells in tooth tissue regeneration—challenges and limitations,” Stem Cell Reviews, vol. 7, no. 3, pp. 683–692, 2011.
[3]
E. K. Yim and M. P. Sheetz, “Force-dependent cell signaling in stem cell differentiation,” Stem Cell Research & Therapy, vol. 3, no. 5, article 41, 2012.
[4]
S. Martino, F. D'Angelo, I. Armentano, J. M. Kenny, and A. Orlacchio, “Stem cell-biomaterial interactions for regenerative medicine,” Biotechnology Advances, vol. 30, no. 1, pp. 338–351, 2012.
[5]
F. D'Angelo, I. Armentano, I. Cacciotti, et al., “Tuning multi/pluri-potent stem cell fate by electrospun poly(L-lactic acid)-calcium-deficient hydroxyapatite nanocomposite mats,” Biomacromolecules, vol. 13, no. 5, pp. 1350–1360, 2012.
[6]
F. D'Angelo, I. Armentano, S. Mattioli et al., “Micropatterned hydrogenated amorphous carbon guides mesenchymal stem cells towards neuronal differentiation,” European Cells & Materials, vol. 20, pp. 231–244, 2010.
[7]
E. Lizundia, J. R. Sarasua, F. D'Angelo, et al., “Biocompatible Poly (L-lactide) / MWCNT nanocomposites: morphological characterization, electrical properties and stem cell interaction,” Macromolecular Bioscience, vol. 12, no. 7, pp. 870–881, 2012.
[8]
A. Bianco, C. Del Gaudio, S. Baiguera et al., “Microstructure and cytocompatibility of electrospun nanocomposites based on poly(ε-caprolactone) and carbon nanostructures,” International Journal of Artificial Organs, vol. 33, no. 5, pp. 271–282, 2010.
[9]
M. Dottori, C. Tay, and S. M. Hughes, “Neural development in human embryonic stem cells-applications of lentiviral vectors,” Journal of Cellular Biochemistry, vol. 112, no. 8, pp. 1955–1962, 2011.
[10]
I. Armentano, L. Marinucci, M. Dottori et al., “Novel poly(L-lactide) PLLA/SWNTs nanocomposites for biomedical applications: material characterization and biocompatibility evaluation,” Journal of Biomaterials Science, Polymer Edition, vol. 22, no. 4–6, pp. 541–556, 2011.
[11]
E. M. Cronin, F. A. Thurmond, R. Bassel-Duby et al., “Protein-coated poly(L-lactic acid) fibers provide a substrate for differentiation of human skeletal muscle cells,” Journal of Biomedical Materials Research A, vol. 69, no. 3, pp. 373–381, 2004.
[12]
S. Sun, I. Titushkin, and M. Cho, “Regulation of mesenchymal stem cell adhesion and orientation in 3D collagen scaffold by electrical stimulus,” Bioelectrochemistry, vol. 69, no. 2, pp. 133–141, 2006.
[13]
D. Yaffe and O. Saxel, “Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle,” Nature, vol. 270, no. 5639, pp. 725–727, 1977.
[14]
S. T. Cooper, A. L. Maxwell, E. Kizana et al., “C2C12 co-culture on a fibroblast substratum enables sustained survival of contractile, highly differentiated myotubes with peripheral nuclei and adult fast myosin expression,” Cell Motility and the Cytoskeleton, vol. 58, no. 3, pp. 200–211, 2004.
[15]
A. Consiglio, S. Martino, D. Dolcetta et al., “Metabolic correction in oligodendrocytes derived from metachromatic leukodystrophy mouse model by using encapsulated recombinant myoblasts,” Journal of the Neurological Sciences, vol. 255, no. 1-2, pp. 7–16, 2007.
[16]
S. Martino, A. Consiglio, C. Cavalieri et al., “Expression and purification of a human, soluble Arylsulfatase a for Metachromatic Leukodystrophy enzyme replacement therapy,” Journal of Biotechnology, vol. 117, no. 3, pp. 243–251, 2005.
[17]
S. Martino, F. D'Angelo, I. Armentano et al., “Hydrogenated amorphous carbon nanopatterned film designs drive human bone marrow mesenchymal stem cell cytoskeleton architecture,” Tissue Engineering A, vol. 15, no. 10, pp. 3139–3149, 2009.
[18]
E. K. F. Yim and K. W. Leong, “Significance of synthetic nanostructures in dictating cellular response,” Nanomedicine, vol. 1, no. 1, pp. 10–21, 2005.
[19]
P.-Y. Wang, H.-T. Yu, and W.-B. Tsai, “Modulation of alignment and differentiation of skeletal myoblasts by submicron ridges/grooves surface structure,” Biotechnology and Bioengineering, vol. 106, no. 2, pp. 285–294, 2010.
[20]
N. Wedhas, H. J. Klamut, C. Dogra, A. K. Srivastava, S. Mohan, and A. Kumar, “Inhibition of mechanosensitive cation channels inhibits myogenic differentiation by suppressing the expression of myogenic regulatory factors and capcase-3 activity,” FASEB Journal, vol. 19, no. 14, pp. 1986–1997, 2005.
[21]
E. Lizundia, A. Oleaga, A. Salazar, and J. R. Sarasua, “Nano- and microstructural effects on thermal properties of poly (l-lactide)/multi-wall carbon nanotube composites,” Polymer, vol. 53, no. 12, pp. 2412–2421, 2012.
[22]
K. Kolind, K. W. Leong, and F. Besenbacher M, “Guidance of stem cell fate on 2D patterned surfaces,” Biomaterials, vol. 33, no. 28, pp. 6626–6633, 2012.
[23]
S. F. Badylak, D. J. Weiss, A. Caplan, and P. MacChiarini, “Engineered whole organs and complex tissues,” The Lancet, vol. 379, no. 9819, pp. 943–952, 2012.
[24]
P. Clark, G. A. Dunn, A. Knibbs, and M. Peckham, “Alignment of myoblasts on ultrafine gratings inhibits fusion in vitro,” International Journal of Biochemistry and Cell Biology, vol. 34, no. 7, pp. 816–825, 2002.
[25]
E. N. Olson, “Interplay between proliferation and differentiation within the myogenic lineage,” Developmental Biology, vol. 154, no. 2, pp. 261–272, 1992.
[26]
S. Tavakol, M. R. Nikpour, and A. Amani, “Bone regeneration based on nano-hydroxyapatite, and hydroxyapatite/chitosan nanocomposites: an in vitro, and in vivo comparative study,” Journal of Nanoparticle Research, vol. 15, pp. 1373–1378, 2013.
[27]
D. Petrá?ková, K. Buriánková, E. Tesa?ová, et al., “Surface hydrophobicity and roughness influences the morphology and biochemistry of streptomycetes during attached growth and differentiation,” FEMS Microbiology Letters, vol. 342, no. 2, pp. 147–156, 2013.
[28]
M. O. Klein, A. Bijelic, T. Ziebart et al., “Submicron scale-structured hydrophilic titanium surfaces promote early osteogenic gene response for cell adhesion and cell differentiation,” Clinical Implant Dentistry and Related Research, vol. 15, no. 2, pp. 166–175, 2011.