Table 1.
Primary antibodies.
Table 2.
Secondary antibodies.
Table 3.
Primer and gene information for qPCR.
Fig 1.
Differential preservation of collagen IV and fibronectin in sections treated with trypsin, SDS and PLA2.
Quadriceps muscle sections treated with trypsin (b, h), SDS (d, j) and PLA2 (f, l), and untreated controls (a, c, e, g, i, k), were fixed with 4% paraformaldehyde and stained with rabbit polyclonal antibodies against collagen IV and fibronectin followed by a goat anti-rabbit FITC conjugated secondary antibody. Nuclei are stained with DAPI (blue). Arrows indicate nuclear material remaining after decellularisation in SDS treated samples. Scale bar—100 μm.
Fig 2.
ECM components are preserved in PLA2 decellularised muscle sections.
Untreated (a–d, i–k) and PLA2 decellularised (e–h, l–n) sections were immunostained. The antibodies against collagen I (a, e), collagen III (b, f), collagen VI (c, g) and perlecan (k, n) were rabbit polyclonals and the second antibody was goat anti-rabbit FITC. Antibodies against laminin α2 (d, h) and decorin (j, m) were rat and sheep polyclonals respectively, and secondary antibodies were goat anti-rat and donkey anti-sheep FITC. The mAb, EV3C3, (i, l) recognises a heparan sulfate epitope [46] and contains a vesicular stomatitis virus Glycoprotein (VSV-G) tag. A rabbbit anti-VSV antibody followed by goat-anti-rabbit FITC was used. Nuclei are stained with DAPI (blue). Scale bars—100 μm for a-h, and 50 μm for i-n.
Fig 3.
SDS-PAGE and Western blotting indicates preservation of matrix proteins.
(A) After PLA2 decellularisation ECM proteins were solubilised, resolved on a 7.5% SDS-PAGE and visualized by Coomassie Blue staining: PLA2 decellularised matrix (lane 3) and collagen 1 (lane 2). (B) Muscle extracts (untreated and PLA2 treated) were resolved on 4–15% gradient gels and membranes were probed with antibodies against collagen I, collagen VI, fibronectin, and perlecan (all rabbit polyclonals) and laminin α2 (rat, clone 4H8-2). Secondary antibodies were anti-rabbit or anti-rat conjugated to HRP, and blots were visualized using ECL. C: untreated muscle extract, PLA2: decellularise muscle extract.
Fig 4.
Structural integrity of decellularized muscle tissue.
Scanning electron micrographs of 0.5 mm thick rat muscle sections (a, b) and 60 μm thick murine muscle sections (c). Images are representative of five samples. Scale bars are 10 μm (a–b) and 20 μm (c).
Fig 5.
ECM proteins support C2C12 myoblast proliferation in serum free cultures.
(A) Phase contrast images show C2C12 myoblasts cultured on collagen I (a, d), fibronectin (b, e) and solubilized muscle matrix (c, f) after 1 and 4 days in serum free culture. Scale bars are 150 μm. A section of “f” is shown at a higher magnification (Scale bar = 200 μm). Proliferation of C2C12 cells on protein substrates in serum free medium was assessed by a direct cell count (g). Data are mean ± SD of 4 independent experiments.
Fig 6.
Immunofluorescent staining of mature C2C12 myotubes.
C2C12 myoblasts were cultured for 4 and 6 days in differentiation medium on collagen I (a–c), fibronectin (d–f) and muscle matrix (g–i), and visualized using the mouse anti-MyHCB mAb (clone NOQ7.5.4D) and goat anti-mouse AF488. Arrows indicate striations (c, f, g and i). Nuclei are stained with DAPI (blue). Scale bars are 50 μm for a–b, d–e, g–h and g insert; and 25 μm for c, f and i. Myotube width was measured using ImageJ software (j), values are the mean and SD of 50 myotubes.
Fig 7.
Quantitative Real time PCR analysis reveals muscle differentiation on matrix proteins.
qPCR was used to assess the expression of markers (Table 3) in differentiating C2C12 cells grown in serum free medium on collagen I (a) fibronectin (b) and solubilised muscle matrix (c) at days 1, 4 and 8 of differentiation. Relative expression levels for MYF5, MYOG, MYH1, MYH3, MYH7, ACTA1 and ACTC1 were normalized to the Ct value of the reference gene (SDHA) and fold change was determined using the 2-delta delta Ct method. Day 4 and Day 8 expression levels were normalized to Day 1 and log10 transformed. Mean ± SE of 4 biological replicates are shown. Significance was determined using a two-tailed t test at P<0.05*.
Fig 8.
Decellularised 3D muscle scaffolds supported proliferation and migration of C2C12 cells.
The click-iT EdU assay was performed on C2C12 myoblasts cultured for 3 days in proliferation media (proliferating cells: green, other cells: blue; scale bar 100 μm) (a), colour-coded image of the distribution of cells in the scaffold after 3 days (b). The EdU assay was performed on cells cultured for 3 days in proliferation media and 6 days in differentiation media (cells coloured as above; scale bar—100 μm)(c). Cells were grown for 7 days in proliferation media, stained with Kwik Diff (nuclei—red; cytoplasm and plasma membrane—blue) and imaged using bright field microscopy (Zeiss Axioskop), scale bar-100 μm (d–f). The arrows indicate C2C12 myoblasts aligning on longitudinal (e) or circular (f) tracks of ECM.
Fig 9.
Secretion and orientation of matrix proteins by C2C12 cells under serum free conditions on etched glass.
(a) C2C12 cells (3500/cm2) were seeded on un-etched and etched glass coverslips in the wells of a 12-well plate and cultured for 3 days in serum free proliferation medium. After 3 days, the media was changed to a serum free differentiation medium and cells were cultured for another 6 days. Post differentiation, cells were fixed and stained with anti-myosin mouse monoclonal (NOQ7.5.4D) antibody. The secondary antibody used was a goat anti-mouse alexafluor 488 antibody. Scale bar—100 μm. (b) The expression of matrix proteins by C2C12 myoblasts was examined on etched coverslips 3.5 hours after plating and in (c) undifferentiated (day 3 proliferation) and differentiated (day 6 differentiation) cell cultures. At these time-points cells were fixed and the coverslips immunostained using antibodies recognising perlecan, collagen I, collagen IV and fibronectin (all rabbit polyclonal antibodies.) The secondary antibody was goat anti-rabbit alexafluor 488 antibody. Nuclei are stained with DAPI (blue). Scale bar—50 μm.