Minimally invasive approach to the repair of injured skeletal muscle with a shape-memory scaffold

Abstract

Repair of injured skeletal muscle by cell therapies has been limited by poor survival of injected cells. Use of a carrier scaffold delivering cells locally, may enhance in vivo cell survival, and promote skeletal muscle regeneration. Biomaterial scaffolds are often implanted into muscle tissue through invasive surgeries, which can result in trauma that delays healing. Minimally invasive approaches to scaffold implantation are thought to minimize these adverse effects. This hypothesis was addressed in the context of a severe mouse skeletal muscle injury model. A degradable, shape-memory alginate scaffold that was highly porous and compressible was delivered by minimally invasive surgical techniques to injured tibialis anterior muscle. The scaffold controlled was quickly rehydrated in situ with autologous myoblasts and growth factors (either insulin-like growth factor-1 (IGF-1) alone or IGF-1 with vascular endothelial growth factor (VEGF)). The implanted scaffolds delivering myoblasts and IGF-1 significantly reduced scar formation, enhanced cell engraftment, and improved muscle contractile function. The addition of VEGF to the scaffold further improved functional recovery likely through increased angiogenesis. Thus, the delivery of myoblasts and dual local release of VEGF and IGF-1 from degradable scaffolds implanted through a minimally invasive procedure effectively promoted the functional regeneration of injured skeletal muscle.

Figures

Figure 1

Experimental flow chart and a schematic for the minimally invasive delivery of the scaffold. (a) The experimental flow chart. (b) Photograph of original macroporous alginate scaffold (16.5 × 2.6 × 0.12 mm) with scanning electron microscopy image (left). Scaffolds had an average pore diameter of 400 µm. Scaffolds were loaded into syringe for delivery via catheter. The middle photograph demonstrates the syringe-needle-catheter device containing a solution of cells and growth factors after the scaffold has been injected; a scaffold (16.5 × 2.6 × 1.1 mm) is also shown following injection and rehydration with cells and growth factors, along with a higher magnification photograph. Scaffolds were implanted subcutaneously along the tibialis anterior via a minimally invasive surgery. Scale bars are shown on images. IGF-1, insulin-like growth factor-1; VEGF, vascular endothelial growth factor.

Figure 2

Masson's trichrome staining for fibrotic tissue in longitudinal sections of explanted tibialis anterior muscles at week 6 after the treatments. (a) Injury only (controls). Collagen deposition/fibrotic tissue was stained blue (dashed black lines), while the muscle tissue was stained in red. (b) Cells/IGF-1 (no scaffold). (c) Cells/IGF-1/scaffold. (d) Cells/IGF-1/VEGF/scaffold. (e) Quantification of fibrotic tissue area in different treatment groups. The fibrotic tissue area was measured in the Masson's trichrome-stained images 2 and 6 weeks after the treatments, and expressed as the percent of fibrotic tissue area/whole tissue area. The unoperated controls had fibrotic areas of less than 5% (data not shown). Bars represent the mean ± SEM. *P < 0.05 (n = 9–12). IGF-1, insulin-like growth factor-1; VEGF, vascular endothelial growth factor.

Figure 3

The recovery of muscle force and muscle weight after the treatments. (a) Quantification of force of the tibialis anterior (TA) muscles in the different groups at week 2 and week 6 after the treatments. The muscle forces were normalized to the wet weight of the corresponding muscles, and the force/weight ratio was used to indicate recovery of muscle function. Bars represent the mean ± SEM. *P < 0.05; **P < 0.01 (n = 5–8 per treatment group). (b) Quantification of TA muscle wet weight in the different groups at week 2 and week 6 after the treatments (n = 9–12 per treatment group). Bars represent the mean ± SEM. *P < 0.05; n.s., not significant. IGF-1, insulin-like growth factor-1; VEGF, vascular endothelial growth factor.

Figure 4

Endothelial cell density in the muscle tissue. At week 6 after the treatments, the average number of vascular endothelial cells (CD31 staining positive, see “Materials and methods” for details) per 104 µm2 tissue area was counted. Bars represent the mean ± SEM. *P < 0.05; **P < 0.01 (n = 9–12). IGF-1, insulin-like growth factor-1; VEGF, vascular endothelial growth factor.

Figure 5

Myofiber diameters at week 6 after the treatments. Muscle fiber cross-sectional areas were measured with ImageJ software. The fiber diameters were calculated from the area measurements. Bars represent the mean ± SEM of each group (n = 9–12). **P < 0.01. IGF-1, insulin-like growth factor-1; VEGF, vascular endothelial growth factor.

Figure 6

Presence of centrally located cell nuclei in the different groups at week 6 after the treatments. (a) Unoperated controls. (b) Cells/IGF-1/VEGF/scaffold. (c and d) Enlarged views of the areas indicated by the dashed lines in (a) and (b), respectively. Nuclei were indicated by white arrows. (e) Quantification of centrally located nuclei in different groups. Bars represent the mean ± SEM. *P < 0.05; **P < 0.01 (n = 9–12). IGF-1, insulin-like growth factor-1; VEGF, vascular endothelial growth factor.

Figure 7

Integration of myoblasts into host muscle tissues. (a–d) Representative images of green flourescent protein (GFP) staining of tibialis anterior (TA) muscle cross-sections at week 6 after the treatments. GFP staining (pseudored) was merged with the nuclei staining (blue). (a) Injury only; (b) cells/IGF-1; (c) cells/IGF-1/scaffold; (d) cells/IGF-1/VEGF/scaffold. (e–h) Enlarged views of the areas outlined by the white dashed lines in a–d, respectively; individual myofibers outlined with dashed white lines in (g) and (h). Positive GFP staining was indicated with arrowheads. (i–l) The corresponding spectral representation (heat map) of the fluorescence intensity of GFP staining in e–h. (m) Quantification of the percentage of GFP-positive myofibers after the treatments. Bars represent the mean ± SEM. ***P < 0.001 (five random fields per slide, three slides per treatment). n.a., not applicable; n.s., not significant. IGF-1, insulin-like growth factor-1; VEGF, vascular endothelial growth factor.