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. 2017 Nov;11(11):3178-3192.
doi: 10.1002/term.2227. Epub 2016 Nov 22.

Silk Fibroin Scaffolds With Muscle-Like Elasticity Support in Vitro Differentiation of Human Skeletal Muscle Cells

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Free PMC article

Silk Fibroin Scaffolds With Muscle-Like Elasticity Support in Vitro Differentiation of Human Skeletal Muscle Cells

Vishal Chaturvedi et al. J Tissue Eng Regen Med. .
Free PMC article

Abstract

Human adult skeletal muscle has a limited ability to regenerate after injury and therapeutic options for volumetric muscle loss are few. Technologies to enhance regeneration of tissues generally rely upon bioscaffolds to mimic aspects of the tissue extracellular matrix (ECM). In the present study, silk fibroins from four Lepidoptera (silkworm) species engineered into three-dimensional scaffolds were examined for their ability to support the differentiation of primary human skeletal muscle myoblasts. Human skeletal muscle myoblasts (HSMMs) adhered, spread and deposited extensive ECM on all the scaffolds, but immunofluorescence and quantitative polymerase chain reaction analysis of gene expression revealed that myotube formation occurred differently on the various scaffolds. Bombyx mori fibroin scaffolds supported formation of long, well-aligned myotubes, whereas on Antheraea mylitta fibroin scaffolds the myotubes were thicker and shorter. Myotubes were oriented in two perpendicular layers on Antheraea assamensis scaffolds, and scaffolds of Philosamia/Samia ricini (S. ricini) fibroin poorly supported myotube formation. These differences were not caused by fibroin composition per se, as HSMMs adhered to, proliferated on and formed striated myotubes on all four fibroins presented as two-dimensional fibroin films. The Young's modulus of A. mylitta and B. mori scaffolds mimicked that of normal skeletal muscle, but A. assamensis and S. ricini scaffolds were more flexible. The present study demonstrates that although myoblasts deposit matrix onto fibroin scaffolds and create a permissive environment for cell proliferation, a scaffold elasticity resembling that of normal muscle is required for optimal myotube length, alignment, and maturation. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd. StartCopTextStartCopText© 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

Keywords: biomaterials; elasticity; extracellular matrix; myotubes; primary human myoblasts; silk fibroin; skeletal muscle tissue engineering.

Figures

Figure 1
Figure 1
Schematic representation of silk protein fibroin isolation from mulberry and non‐mulberry silkworm species and the preparation of three‐dimensional (3D) fibroin scaffolds. SDS, sodium dodecyl sulphate. [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 2
Figure 2
Human skeletal muscle myoblasts (HSMMs) adhere and secrete extracellular matrix (ECM) proteins on three‐dimensional (3D) silk scaffolds. The HSMMs were cultured on 3D silk scaffolds for 2 days in skeletal muscle growth medium‐2 proliferation medium, then fixed and stained with rhodamine–phalloidin (F‐actin staining; b,f,j,n), or antibodies recognizing either fibronectin (c,g,k,o), or perlecan (d,h,l,p). 4′,6‐diamidino‐2‐phenylindole (DAPI) stained the silk scaffolds (all panels). Images were captured using a Nikon A1 confocal laser scanning microscope. The merged images of several z‐stack images (5 μm each stack, 150–200 μm deep into the scaffold) are presented. A representative image of six fields of view is shown. Bar: 50 μm. [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 3
Figure 3
Immunostained images of differentiated human skeletal muscle myoblasts (HSMMs) on three‐dimensional (3D) silk scaffolds. The HSMMs were cultured on the silk scaffolds for 4 days in proliferation medium and a further 4 days in differentiation medium. After differentiation, the scaffolds were stained with anti‐myosin monoclonal antibody (NOQ7.5.4D) and goat anti‐mouse IgG‐AF488. The nuclei are stained with 4′,6‐diamidino‐2‐phenylindole (DAPI, blue). Images were captured using a Nikon A1 confocal microscope. The 3D merged images of several z‐stack images (5 μm each stack, 150–200 μm deep into scaffold) are represented. Bar: 100 μm. [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 4
Figure 4
Human skeletal muscle myoblasts (HSMMs) form a three‐dimensional (3D) muscle‐like tissue on Bombyx mori scaffolds. The HSMMs were cultured for 4 days in skeletal muscle growth medium‐2 proliferation medium and 4 days in differentiation medium on B. mori silk scaffolds. Myotubes were stained with anti‐myosin monoclonal antibody (NOQ.5.4.D), followed by a goat anti‐mouse AF488 conjugated second antibody. The images were captured using an Ultraview spinning disc confocal microscope (PerkinElmer). To capture a large area and estimate the coverage of the myotubes on the scaffold surface, 50 z‐stack images (2.5 μm each stack) were stitched together using Volocity software. Muscle fibre length (coloured dotted lines and boxed numbers) was measured by tracing individual fibres using Volocity software. The area covered in this image is indicated by the following: x‐axis, 1.58 mm; y‐axis, 1.213 mm; z‐stack, 125 μm. Bar: 150 μm. [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 5
Figure 5
Scanning electron micrographs of three‐dimensional (3D) silk scaffolds with and without human skeletal muscle myoblasts (HSMMs). (A) The scaffolds without cells (a, c, e, g) and at day 5 after seeding with HSMMs (b,d,f,h) are shown. Bars: 100 μm. (B) Highly magnified scanning electron micrographs of Antheraea mylitta silk scaffolds. The blank scaffold (a) shows the interconnected pores. HSMMs are shown at day 2 after seeding with a spread cell (b) indicated by arrow, and (c) matrix proteins deposited onto the scaffold (arrow). Bar: 10 μm (a,b), 2 μm (c)
Figure 6
Figure 6
Young's modulus and viscous modulus of three‐dimensional silk scaffolds. Strain sweep was performed from 0.01–3% at 1 Hz and 37°C. Scaffold dimensions were 14 mm in diameter and 4 mm thick and four replicate scaffolds were measured. Grey bars are Young's modulus (YM), hatched bars are viscous modulus (VM). Data are mean ± standard deviation (n = 4). Am, Antheraea mylitta; Bm, Bombyx mori; Aa, Antheraea assamensis; Sr, Samia ricini. *YM and VM are significantly greater in Am and Bm compared with Aa and Sr (p ≤ 0.01); #YM and VM are significantly greater in Bm than Am (p ≤ 0.01)
Figure 7
Figure 7
Human skeletal muscle myoblasts (HSMMs) are viable on silk fibroin substrates. Metabolic activity of HSMMs on four different silk fibroins, two extracellular matrix proteins and tissue culture plastic in Skeletal Muscle Growth Medium‐2 was assessed using the Alamar Blue assay. The days in culture when the Alamar Blue dye was added and absorbance measured are indicated. The relative fluorescence units (RFU) were calculated relative to the control (media, no cells). Data are mean ± standard deviation (n = 4). Am, Antheraea mylitta; Bm, Bombyx mori; Aa, Antheraea assamensis; Col I, collagen I; Fibro, Fibronectin; Sr, Samia ricini; *p ≤ 0.05
Figure 8
Figure 8
Human skeletal muscle myoblasts (HSMMs) differentiate to form myotubes on silk fibroin substrates. HSMMs were seeded on etched glass coated with the silk fibroins (10 μg/cm2) and cultured for 4 days in skeletal muscle growth medium‐2 proliferation medium and a further 7 or 10 days in differentiation medium. After 7 days of differentiation myotubes were stained with a mouse anti‐myosin heavy chain monoclonal antibody (mAb) (NOQ7.5.4D) and goat anti‐mouse IgG‐AF488 (a–d) (MyHCB, mAb recognizing slow muscle myosin); after 10 days, myotubes were stained with both the anti‐myosin mAb/goat anti‐mouse IgG‐AF488 combination and with rabbit anti‐glucose transporter 4 (GLUT4) antibody followed by goat anti‐rabbit IgG‐AF546 (e–h). The nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI). Myotubes were imaged using a Zeiss Axioskop fluorescent microscope. Bar: 50 μm (a–d), 25 μm (e–h). [Colour figure can be viewed at wileyonlinelibrary.com]
Figure 9
Figure 9
Quantitative real‐time reverse transcription polymerase chain reaction (qRT‐PCR) analysis reveals muscle differentiation on Bombyx mori and Antheraea mylitta three‐dimensional (3D) silk scaffolds. qRT‐PCR was used to determine the expression of five differentiation markers (Table 2) in HSMMs grown in skeletal muscle growth medium‐2 on Bombyx mori (a,c) and A mylitta (b,d) scaffolds at day 4 (a,b) and day 10 (c,d) of differentiation. Relative expression levels for MYF5, MYOD1, MYH1, MYH7 and ACTA1 are normalized to the Ct value of the reference gene (TBP) and fold‐change was determined using the using 2‐delta delta Ct method. Day 4 and day 10 differentiation expression levels were normalized to day 0 and day 2 differentiation, respectively. The mean ± standard error of four biological replicates are presented. The Mann–Whitney U‐test was performed and significance (p) values shown at each time‐point for each gene using *p < 0.05

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