Anchorage of muscle cells to the extracellular matrix is crucial for a range of fundamental biological processes including migration, survival and differentiation. Three-dimensional (3D) culture has been proposed to provide a more physiological in vitro model of muscle growth and differentiation than routine 2D cultures. However, muscle cell adhesion and cell-matrix interplay of engineered muscle tissue remain to be determined. We have characterized cell-matrix interactions in 3D muscle culture and analyzed their consequences on cell differentiation. Human myoblasts were embedded in a fibrin matrix cast between two posts, cultured until confluence, and then induced to differentiate. Myoblasts in 3D aligned along the longitudinal axis of the gel. They displayed actin stress fibers evenly distributed around the nucleus and a cortical mesh of thin actin filaments. Adhesion sites in 3D were smaller in size than in rigid 2D culture but expression of adhesion site proteins, including α5 integrin and vinculin, was higher in 3D compared with 2D (p<0.05). Myoblasts and myotubes in 3D exhibited thicker and ellipsoid nuclei instead of the thin disk-like shape of the nuclei in 2D (p<0.001). Differentiation kinetics were faster in 3D as demonstrated by higher mRNA concentrations of α-actinin and myosin. More important, the elastic modulus of engineered muscle tissues increased significantly from 3.5±0.8 to 7.4±4.7 kPa during proliferation (p<0.05) and reached 12.2±6.0 kPa during differentiation (p<0.05), thus attesting the increase of matrix stiffness during proliferation and differentiation of the myocytes. In conclusion, we reported modulations of the adhesion complexes, the actin cytoskeleton and nuclear shape in 3D compared with routine 2D muscle culture. These findings point to complex interactions between muscle cells and the surrounding matrix with dynamic regulation of the cell-matrix stiffness.
References
[1]
Carmignac V, Durbeej M (2011) Cell-matrix interactions in muscle disease. J Pathol.
[2]
Kanagawa M, Toda T (2006) The genetic and molecular basis of muscular dystrophy: roles of cell-matrix linkage in the pathogenesis. J Hum Genet 51: 915–926.
[3]
Liao H, Zhou GQ (2009) Development and progress of engineering of skeletal muscle tissue. Tissue Eng Part B Rev 15: 319–331.
[4]
Bian W, Bursac N (2009) Engineered skeletal muscle tissue networks with controllable architecture. Biomaterials 30: 1401–1412.
[5]
Orlando G, Baptista P, Birchall M, De Coppi P, Farney A, et al. (2011) Regenerative medicine as applied to solid organ transplantation: current status and future challenges. Transpl Int 24: 223–232.
Shah R, Sinanan ACM, Knowles JC, Hunt NP, Lewis MP (2005) Craniofacial muscle engineering using a 3-dimensional phosphate glass fibre construct. Biomaterials 26: 1497–1505.
[8]
Williamson MR, Adams EF, Coombes AGA (2006) Gravity spun polycaprolactone fibres for soft tissue engineering: interaction with fibroblasts and myoblasts in cell culture. Biomaterials 27: 1019–1026.
[9]
Levy-Mishali M, Zoldan J, Levenberg S (2009) Effect of scaffold stiffness on myoblast differentiation. Tissue Eng Part A 15: 935–944.
[10]
Rhim C, Lowell DA, Reedy MC, Slentz DH, Zhang SJ, et al. (2007) Morphology and ultrastructure of differentiating three-dimensional mammalian skeletal muscle in a collagen gel. Muscle & nerve 36: 71–80.
[11]
Janmey PA, Winer JP, Weisel JW (2009) Fibrin gels and their clinical and bioengineering applications. J R Soc Interface 6: 1–10.
[12]
Gerard C, Forest MA, Beauregard G, Skuk D, Tremblay JP (2011) Fibrin gel improves the survival of transplanted myoblasts. Cell Transplant.
[13]
Huang Y-C, Dennis RG, Larkin L, Baar K (2005) Rapid formation of functional muscle in vitro using fibrin gels. Journal of applied physiology (Bethesda, Md : 1985) 98: 706–713.
[14]
Matsumoto T, Sasaki J-i, Alsberg E, Egusa H, Yatani H, et al. (2007) Three-dimensional cell and tissue patterning in a strained fibrin gel system. PloS one 2: e1211.
[15]
Vandenburgh H (2010) High-content drug screening with engineered musculoskeletal tissues. Tissue engineering Part B, Reviews 16: 55–64.
[16]
Vandenburgh H, Shansky J, Benesch-Lee F, Barbata V, Reid J, et al. (2008) Drug-screening platform based on the contractility of tissue-engineered muscle. Muscle & nerve 37: 438–447.
[17]
Engler AJ, Griffin MA, Sen S, Bonnemann CG, Sweeney HL, et al. (2004) Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments. J Cell Biol 166: 877–887.
[18]
Tse JR, Engler AJ (2011) Stiffness gradients mimicking in vivo tissue variation regulate mesenchymal stem cell fate. PLoS One 6: e15978.
[19]
Hansen A, Eder A, Bonstrup M, Flato M, Mewe M, et al. (2010) Development of a drug screening platform based on engineered heart tissue. Circulation research 107: 35–44.
[20]
Sander EA, Barocas VH, Tranquillo RT (2011) Initial fiber alignment pattern alters extracellular matrix synthesis in fibroblast-populated fibrin gel cruciforms and correlates with predicted tension. Ann Biomed Eng 39: 714–729.
[21]
Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: RESEARCH0034.
[22]
Vandenburgh HH, Karlisch P, Farr L (1988) Maintenance of highly contractile tissue-cultured avian skeletal myotubes in collagen gel. In vitro cellular & developmental biology : journal of the Tissue Culture Association 24: 166–174.
[23]
DeQuach JA, Mezzano V, Miglani A, Lange S, Keller GM, et al. (2010) Simple and high yielding method for preparing tissue specific extracellular matrix coatings for cell culture. PLoS One 5: e13039.
[24]
Koning M, Harmsen MC, van Luyn MJ, Werker PM (2009) Current opportunities and challenges in skeletal muscle tissue engineering. J Tissue Eng Regen Med 3: 407–415.
[25]
Schaaf S, Shibamiya A, Mewe M, Eder A, Stohr A, et al. (2011) Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology. PLoS One 6: e26397.
[26]
Dado D, Levenberg S (2009) Cell-scaffold mechanical interplay within engineered tissue. Semin Cell Dev Biol 20: 656–664.
[27]
Winer JP, Oake S, Janmey PA (2009) Non-linear elasticity of extracellular matrices enables contractile cells to communicate local position and orientation. PLoS One 4: e6382.
[28]
Formigli L, Meacci E, Zecchi-Orlandini S, Orlandini GE (2007) Cytoskeletal reorganization in skeletal muscle differentiation: from cell morphology to gene expression. Eur J Histochem 51: Suppl 121–28.
[29]
Hayward LJ, Zhu YY, Schwartz RJ (1988) Cellular localization of muscle and nonmuscle actin mRNAs in chicken primary myogenic cultures: the induction of alpha-skeletal actin mRNA is regulated independently of alpha-cardiac actin gene expression. J Cell Biol 106: 2077–2086.
[30]
Barczyk M, Carracedo S, Gullberg D (2010) Integrins. Cell Tissue Res 339: 269–280.
[31]
Moore SW, Roca-Cusachs P, Sheetz MP (2010) Stretchy proteins on stretchy substrates: the important elements of integrin-mediated rigidity sensing. Dev Cell 19: 194–206.
[32]
Burridge K, Fath K, Kelly T, Nuckolls G, Turner C (1988) Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol 4: 487–525.
[33]
Hakkinen KM, Harunaga JS, Doyle AD, Yamada KM (2011) Direct comparisons of the morphology, migration, cell adhesions, and actin cytoskeleton of fibroblasts in four different three-dimensional extracellular matrices. Tissue Eng Part A 17: 713–724.
[34]
Kubow KE, Horwitz AR (2011) Reducing background fluorescence reveals adhesions in 3D matrices. Nat Cell Biol 13: 3–5.
Fraley SI, Feng Y, Krishnamurthy R, Kim D-H, Celedon A, et al. (2010) A distinctive role for focal adhesion proteins in three-dimensional cell motility. Nature Cell Biol 12: 598–604.
[38]
Engel A, Franzini-Armstrong C (2004) Myology. New-York: The McGraw-Hill Companies.
[39]
Simon DN, Wilson KL (2011) The nucleoskeleton as a genome-associated dynamic ‘network of networks’. Nat Rev Mol Cell Biol 12: 695–708.
[40]
Khatau SB, Hale CM, Stewart-Hutchinson PJ, Patel MS, Stewart CL, et al. (2009) A perinuclear actin cap regulates nuclear shape. Proc Natl Acad Sci U S A 106: 19017–19022.
