Fig. 2: Highly crosslinked, stiff myoscaffolds are resistant to SMPC-mediated remodeling.

a Indirect immunofluorescence confocal microscopy of SMPCs (CDMD 1002 cells) cultured for 5 days on WT and mdx myoscaffolds (myoscaffold + cells). A decellularized section from each group was not seeded with SMPCs and served as a control (myoscaffold). Sections were stained with antibodies recognizing collagen I (Col I) (green), along with phalloidin as an actin cytoskeleton marker (red) and DAPI as a nuclear marker (blue) (observations from n = 5 independent experiments). Scale bars, 25 μm (×20), and 8 μm (×63). b Graph showing values for the remodeling index (RI: pre/post-remodeling pixel intensity) from WT and mdx myoscaffolds stained for Col I (CDMD 1002 cells, n = 20 endomysial locations/×20 image, results are mean ± s.d.; based on observations from n = 3 independent experiments). P values reflect analysis by two-tailed unpaired t-test. c Individual tiles from stacked confocal images of SMPCs (CDMD 1002 cells) cultured on WT and mdx myoscaffolds. Sections were stained for collagen I (green), phalloidin (red), and DAPI (blue). SMPCs cultured on WT myoscaffolds remodeled and integrated into the ECM, visualized as cells extending into the endomysium and through the myoscaffold thickness. SMPCs cultured on mdx myoscaffolds did not integrate into the myoscaffold, localizing adjacent to the basement membrane or on top of the ECM (observations from n = 5 independent experiments). Scale bar, 25 μm. d, e Quantification of immature dihydroxy lysinonorleucine (DHLNL) and dehydrohydroxy-lysinonorleucine (dHLNL) crosslinks (d), and mature hydroxylyslpyridinoline (HL) crosslinks (e) in WT (n = 5) and mdx (n = 5) quadriceps samples. Values are expressed as the Log2 normalized peak area (NPA) (mean ± s.d.). P values reflect analysis by two-tailed unpaired t-test. f Images of WT and mdx myoscaffolds under the AFM probe during testing and sample traces from the force-separation curves collected from one location on each sample. g Graph showing the Young’s modulus values acquired from a 20 ×20 μm area of the endomysium of a WT myoscaffold (1 biologic sample, n = 154 points), along with a mdx region without evidence of scars (mdx^NS)(1 biologic sample, n = 256 points) and a mdx region with fibrotic scars (mdx^S)(1 biologic sample, n = 240 points) in mdx myoscaffolds. Graph showing mean ± s.d. (n = 3 independent experiments). P values reflect analysis by one-way ANOVA. h Graph showing the average Young’s modulus values (mean ± s.d.) collected from 20 × 20 μm areas from WT (n = 3 biologic samples, average of 67–154 points/sample) and mdx^S (n = 3 biologic samples, average of 111–256 points/sample) myoscaffolds. P values reflect analysis by two-tailed unpaired t-test.