In vivo generation of a mature and functional artificial skeletal muscle. (2015)
Figure 4

PF-embedded Mabs grafted in an ablated TA injury, showing full recovery of muscular morphology and functionality

A, B Gross morphology of the TA injury at 6 months after massive muscle ablation, revealing the new artificial TA regeneration (arrow) when grafted with PF-embedded Mabs-PlGF (B) and absence of TA muscle (arrow) when treated with empty PF as a control (A).

C PF-embedded Mabs-PlGF-derived artificial TA (right) showing comparable size to the wild-type normal TA (left).

D–O Immunofluorescence against myosin heavy chain (MyHC) (red) and laminin (green) on mice TA cross sections at early (10 days) (D–I) and later stage (6 months) (J–O) after massive removal of the muscle (D, G, J, M are the results of a collage of several photos). TA regeneration at early stages showed very few residual myofibres after implantation of acellular PF in place of the ablated TA (white box in D), an enlarged view (E), and DAPI-counterstained view (F) reveals the infiltration of undifferentiated mono-nucleated cells. Conversely, a PF-embedded Mabs-PlGF graft (G) demonstrated early-stage regeneration with several MyHC-positive (red) regenerating myofibres surrounded by laminin (green) (H), most of these (arrows) being centre-nucleated (DAPI labelled in I). At the later stages, acellular PF implanted mice (J) showed a very small portion of muscle tissue likely derived from residual myofibres from TA removal, higher magnification (K) revealed very few mature myofibres with peripheral nuclei (DAPI labelled in L). Contrary, the PF-embedded Mabs-PlGF treatment (M) showed a complete replacement of the ablated TA with the recovered native structure of MyHC-positive myofibres surrounded by laminin (N), some of these (arrows) still being centre nucleate (DAPI labelled in O). Scale bar: (D, G, J, M) 500 μm, (E, F, H, I, K, L, N, O) 100 μm.

P–R Functional analysis demonstrated the effective ability of the PF and Mabs-PlGF combination to generate a complete and functional artificial TA replacing the ablated TA injury. (P) The fore-hind limb grip test diagram reveals functional grab force recovery for mice treated with PF-embedded Mabs-PlGF (TA ABL-PF+MABS) after only 4 weeks post-TA injury, being comparable with not-ablated wild-type (WT) mice and significantly higher than control ablated mice (TA ABL-CONT) or mice implanted with void PF (TA ABL-PF CONT) (n = 10 mice per group). (Q, R) The treadmill analysis of the treated mice relative to wild-type not-ablated (wt) shows both exhaustion time and distance indicative of a full recovery of motor functionality already at 3 weeks after TA ablation with an increase in time and distance run for mice treated with PF-embedded Mabs-PlGF, while control mice (TA ABL-CONT or TA ABL-PF CONT) showed a much lower resistance (n = 10 mice per group). The values shown in the diagrams are expressed as means ± standard error and statistical significance was tested using Student's t-test (P < 0.05 was considered significant).

S–Z Morphological analysis demonstrated the effective ability of the PF and Mabs-PlGF combination to generate a complete and functional artificial TA replacing the ablated TA injury. H&E staining of TA injury in mice by cross section at an early (10 days) (S–V) and late (6 months) (W–Z) time point after ablation (in S, U, W, Y displayed items are obtained from a multi-photo collage). At the early time point, the acellular PF-treated mice revealed few mature muscle fibres that survive the massive ablation (box in S) signs of continued muscle degeneration (arrows in T), whereas PF-embedded Mabs implanted mice showed remarkable muscle regeneration indicated by centre-nucleated myofibres (arrows in V). At later stages, the control mice grafted with acellular PF showed an almost devoid TA (W) with very few muscle fibres (X), in contrast to PF-embedded Mabs, which exhibited a complete muscle replacement generating an artificial TA (Y), enlarged view (Z) proved the complete and mature organization of the newly formed muscle showing capillary and blood vessel around myofibres (arrows). Scale bar: (S, U, W, Y) 500 μm, (T, V, X, Z) 50 μm.

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