Anisotropic Materials for Skeletal-Muscle-Tissue Engineering - PubMed (original) (raw)
Review
Anisotropic Materials for Skeletal-Muscle-Tissue Engineering
Soumen Jana et al. Adv Mater. 2016 Dec.
Abstract
Repair of damaged skeletal-muscle tissue is limited by the regenerative capacity of the native tissue. Current clinical approaches are not optimal for the treatment of large volumetric skeletal-muscle loss. As an alternative, tissue engineering represents a promising approach for the functional restoration of damaged muscle tissue. A typical tissue-engineering process involves the design and fabrication of a scaffold that closely mimics the native skeletal-muscle extracellular matrix (ECM), allowing organization of cells into a physiologically relevant 3D architecture. In particular, anisotropic materials that mimic the morphology of the native skeletal-muscle ECM, can be fabricated using various biocompatible materials to guide cell alignment, elongation, proliferation, and differentiation into myotubes. Here, an overview of fundamental concepts associated with muscle-tissue engineering and the current status of muscle-tissue-engineering approaches is provided. Recent advances in the development of anisotropic scaffolds with micro- or nanoscale features are reviewed, and how scaffold topographical, mechanical, and biochemical cues correlate to observed cellular function and phenotype development is examined. Finally, some recent developments in both the design and utility of anisotropic materials in skeletal-muscle-tissue engineering are highlighted, along with their potential impact on future research and clinical applications.
Keywords: anisotropic materials; micropatterned substrates; muscles; nanofibers; scaffolds; tissue engineering.
© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Figures
Figure 1
Schematic illustration of anatomic structures and organization of skeletal muscle tissue.
Figure 2
Schematic illustration of skeletal muscle tissue engineering using aligned nanofibrous scaffolds. Effective skeletal muscle tissue engineering requires success at multiple stages which may include progenitor cell isolation, cell proliferation, cell culture on scaffolds in vitro, application of dynamic mechanical stimulation via bioreactor, characterization of myogenic implant quality and ultimately implantation into the patient.
Figure 3
Soft lithography microfabrication techniques to generate patterned scaffolds (cross-sectional view). The related methods include: (a) replica molding, (b) microcontact printing, (c) micromolding and (d) microtransfer printing. Reprinted with permission, Copyright © 2012 IOP Publishing.[71]
Figure 4
SEM imaging of myotubes on aligned micropatterned substrates. (a) SEM image of the micropatterned PDMS substrate with a continuous sinusoidal wave pattern (top view), scale bar: 50 μm. (b) SEM image of the micropatterned PDMS substrate (cross-sectional view), scale bar: 10 μm. (c) Myotubes cultured on the micropatterned substrate. Black arrow indicates longitudinal microfeature direction, scale bar: 40 μm. (d,f) Aligned cytoskeleton and nuclei of cells cultured on micropatterned substrates, scale bar: 100 μm. (e,g) Randomly oriented cytoskeleton and nuclei of cells cultured on flat PDMS substrate, scale bar: 100 μm. Reprinted with permission, Copyright © 2009, 2006 Elsevier Ltd. [68, 72]
Figure 5
Fluorescent imaging of myotubes grown on aligned, patterned collagen gel substrates. (a) Fluorescent images of immunostained myotubes cultured for 2 weeks on micropatterned gel substrates of varying stiffness where intermediate stiffness resulted in myotubes with distinctive skeletal muscle striations (scale bar: 20 μm). (b) Immunostained images of myotubes after 4 weeks of culture on micropatterned gel substrates of varied stiffness (scale bar: 20 μm). (c) Myoblasts seeded in two sequential layers on patterned glass. After 1 week of culture, F-actin and myosin are becoming striated for upper layer myotubes, but are not striated for lower level myotubes (scale bar: 10 μm). (d) After 4 weeks in cultured, striated myotubes represent 85% of the myotube population in the upper layer whereas no myotubes are striated in the lower layer. Reprinted with permission, Copyright © 2004 The Rockefeller University Press [54].
Figure 6
Microfabricated poly(lactic-co-glycolic acid) (PLGA) nanoribbon sheet for generation of hierarchically assembled mouse myoblast structure. (a) Schematic depiction of PLGA nanoribbon sheet microfabrication process. (b) Optical and (c) SEM micrograph (scale bar: 20 μm) depicting macro- and microstructure of nanoribbon sheet, and (d) graphical representation of nanoribbon height. (e) Schematic representation of cell bilayer sheet. (f) Immunostaining images of myotubes expressing myosin (green), F-actin (red) and nuclei (blue) on an anisotropic bilayer (A-bilayer, orthogonal) and an isotropic bilayer (I-bilayer, parallel) (scale bars: 100 μm). Reprinted with permission, Copyright © 2015 Elsevier Ltd.[9]
Figure 7
Schematic illustration of an electrospinning system used to produce aligned or randomly-oriented nanofibers. The system is comprised of a grounded collector, electrically charged spinneret and power supply.
Figure 8
A multipurpose electrospinning system and its modular collector electrodes designed in our laboratory. (a) Photograph of the electrospinning system and (b) modular electrodes of different configurations: (I) plate, (II) pin, (III) stepped conical and (IV) cylindrical electrodes. Aligned nanofibrous (c) membrane (scale: 2 μm), (d) cylindrical, (e) tubular and (f) criss-cross strutures (scale: 1 μm). Partly reprinted with permission, Copyright © 2013 Royal Society of Chemistry [87].
Figure 9
Fluorescent imaging of immunostained focal adhesion and transient receptor potential cation channels-1 (TRPC-1) expressed by myoblasts cultured on substrates of different morphologies including flat film, randomly oriented and aligned nanofibers. (a–c) Images of focal adhesions (red) and actin filaments (green) of myoblasts. (d–f) Images of actin filaments (red) and TRPC-1 (green) confirming stretch-activated cation channel upregulation in cells in presence of aligned nanofibers. Nuclei were stained with DAPI. Scale bars: 20 μm. Reprinted with permission, Copyright © 2008 Springer [90].
Figure 10
Aligned nanofibrous membranes and aligned cylindrical nanofibrous scaffolds prepared for skeletal muscle tissue engineering. (a) Photograph of an aligned fibrous membrane deposited between two plate electrodes by electrospinning, scale bar: 2 cm. (b–c) SEM images of an aligned nanofibrous membrane at (b) low magnification (scale bar: 5 μm), and (c) high magnification (scale bar: 700 nm). (d) Fluorescent image of aligned skeletal muscle cells cultured on aligned nanofiber membranes and immunostained for actin (green) and myosin heavy chain (red), scale bar: 20 μm. (e) Photograph of an aligned nanofibrous cylindrical scaffold deposited between two pin electrodes by electrospinning, scale bar: 4 mm. (f) SEM image of the cylindrical scaffold, scale: 50 μm. (g) SEM image of aligned nanofibers in the cylindrical scaffold, scale bar: 700 nm, inset: macroscale image of a cylindrical scaffold, scale bar: 2 mm. (h) SEM image of aligned myotubes grown on the aligned nanofibrous cylinder, scale bar: 100 μm. (i) Fluorescent image of myotubes grown on the nanofibrous cylindrical scaffold immunostained for actin (green), myosin heavy chain (red) and nuclei (blue), scale bar: 20 μm. Partly reprinted with permission, Copyright © 2012 American Chemical Society [16].
Figure 11
Core-shell scaffolds containing aligned nanofiber yarns. (a) Schematic depiction of core-shell column and sheet scaffolds by combining electrospun, aligned nanofiber yarns (NFYs) and hydrogen shell composed of photocurable poly(ethylene glycol)-co-poly(glycerol sebacate) (PEGS-M). (b) Fluorescence-based visualization of core-shell structure showing aligned, 100 μm diameter nanofiber yarn encapsulated by PEGS-M. (c) C2C12 adhesion and proliferation on aligned NFY within PEGS-M shell. Reprinted with permission, Copyright © 2015 American Chemical Society.[19]
Figure 12
Schematic illustration of fabrication process for aligned microporous tubular chitosan scaffolds via TIPS. (a) Chitosan powder and its chemical structure which contains an -NH2 group. (b) Chitosan gel-solution prepared in an aqueous acid solution. (c) Cylindrical Teflon tube as mold filled with chitosan gel-solution. (d) Centrifugation of chitosan gel-solution to remove voids. (e) Application of a thermal gradient between two ends of the tube, resulting in freezing along the cylindrical axis. (f) Lyophilization of frozen chitosan gel-solution. (g) Resultant cylindrical chitosan scaffold with aligned longitudinal microporous structure (longitudinal view), scale bar: 100 μm. (h) Cross-section of aligned chitosan scaffold showing pore dimensions, scale bar: 100 μm. Reprinted with permission, Copyright © 2013 John Wiley & Sons, Inc. [8].
Figure 13
Collagen-based aligned pore scaffolds for skeletal muscle tissue engineering. (a) H&E staining showing collagen (pink) and myotubes (purple) after 7 days of culture to induce myogenic differentiation. Myotubes express myosin heavy chain (b) and synthesized and deposited laminin (c) (scale bar: 100 μm, inset scale bar: 10 μm). (d) SEM micrograph of collagen scaffold microstructure (scale bar: 100 μm). Lower panel: C2C12-collagen scaffold graft after 2 week implantation in anterior tibial muscle of eGFP transgenic mouse (g: graft, h: host, k: knee, arrow: regenerated tendon) (scale bar: 200 μm). Reprinted with permission, Copyright © 2008 Blackwell Publishing Ltd.[13]
Figure 14
Chitosan scaffolds with unidirectional microtubular pores. (a–d) SEM micrographs depicting cross-sectional pore structure and arrangement of 4, 6, 8 and 12 wt% chitosan scaffolds prepared under a medium temperature (MT) gradient during TIPS processing (scale bar: 40 μm). (e–h) SEM micrographs of myotubes formed along the longitudinal direction of microtubular pores formed under the MT-gradient where scaffold is colored green for easier identification of myotubes (scale bar: 60 μm). (i–l) Immunoflurescent staining of C2C12-derived myotubes formed on 8 wt% scaffolds prepared under the MT-gradient, depicting cell nuclei, actin and myosin heavy chain expression after 2 weeks of culture (scale bar: 50 μm). Reprinted with permission, Copyright © 2013 John Wiley & Sons, Inc.[8]
Figure 15
Skeletal muscle tissue engineering scaffold fabricated using an integrated tissue-organ printer (ITOP). (a) Schematic depiction of construct designed to mimic array of aligned muscle fiber bundles including PCL support pillars (green). (b) 3D printed scaffold before (left) and after (right) removing sacrificial Pluronic F-127 hydrogel. (c) Live/dead staining of cells within hydrogel bundles shows high cell viability immediately after printing. (d) Immunofluorescent staining for myosin heavy chain shows myoblast alignment along the fiber after 7 days of culture. (e) Subcutaneous placement of construct in mouse with dissected common peroneal nerve inserted into construct to promote nerve integration. (f) After 2 weeks of implantation, cells within the construct organized into muscle fiber structures as evidenced by desmin staining and (g) expression of myosin heavy chain and α-BTX. (h) Evidence of nerve integration via doublestaining for α-BTX and neurofilament (NF). (i) Constructs became vascularized as evidenced by expression of von Willebrand factor. Reprinted with permission, Copyright © 2016 Nature Publishing Group.[12]
Figure 16
SEM images of cells spreading on anisotropic substrates after 1, 2, and 4 h of culture. (a)–(d) Spreading of a cell on a micropatterned substrate with passage of time, the image in (b) is the magnified image of region indicated in image (a), inset image in (d) is the magnified image of region indicated in image (d). (e–h) Spreading of a cell on an aligned nanofiber-coated film substrate with passage of time, the image in (f) is magnified image of region indicated in image (e), arrow in image (h) shows the alignment of the cell along the nanofibers. The short and long arrows indicate filopodia and lamellipodia, respectively. Scale bars: (a) and (c) 5 μm; (b) 3 μm; (d) and (h) 50 μm; (e) and (g) 20 μm; (f) 10 μm and inset in (d) 5 μm. Reprinted with permission, Copyright © 2015 AVS Publication, Inc.[109]
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