The role of multifunctional delivery scaffold in the ability of cultured myoblasts to promote muscle regeneration

Abstract

Many cell types of therapeutic interest, including myoblasts, exhibit reduced engraftment if cultured prior to transplantation. This study investigated whether polymeric scaffolds that direct cultured myoblasts to migrate outwards and repopulate the host damaged tissue, in concert with release of angiogenic factors designed to enhance revascularizaton of the regenerating tissue, would enhance the efficacy of this cell therapy and lead to functional muscle regeneration. This was investigated in the context of a severe injury to skeletal muscle tissue involving both myotoxin-mediated direct damage and induction of regional ischemia. Local and sustained release of VEGF and IGF-1 from macroporous scaffolds used to transplant and disperse cultured myogenic cells significantly enhanced their engraftment, limited fibrosis, and accelerated the regenerative process. This resulted in increased muscle mass and, improved contractile function. These results demonstrate the importance of finely controlling the microenvironment of transplanted cells in the treatment of severe muscle damage.

Figure 1. Schematic representation of the approach

Engineered scaffold containing transplanted cells and growth factors is able to guide tissue regeneration in situ.

Figure 2. Experimental design

Wild type C57BL/6J mice were subjected to myotoxin injection, and induction of hindlimb ischemia after 6 days by femoral artery ligation. (ab) Tibialis cross-sections, H&E stained before (a) and after (b) muscle injury. (c) Primary GFP myoblasts (green), isolated from transgenic Tg(ACTbEGFP)1Osb, constitutively expressing GFP in all the cells, were seeded in macroporous RGD-modified alginate scaffold (blue) encapsulating VEGF (red) and IGF-1(yellow). (d) Photograph of the macroporous square-shaped alginate scaffold (5 × 5 × 2 mm) in a lyophilized form. (e) Photograph of scaffold adherent to tibialis muscle at 2 weeks time-point.

Figure 3. Donor myoblast engraftment

Representative images of 3 day post-treatment control limbs (A, C) and injured limbs (B, D) treated with scaffolds (B) or bolus injections (D) delivering cells and VEGF/IGF, showing transplanted myoblast contribution to muscle regeneration. GFP expression (green) by immunofluorescence, in transverse sections of muscle harvested at 3 days after transplantation. (E) Quantification of GFP fibers at 3 days after transplantations, normalized to the total cross-sectional area of each muscle. Values are mean ± SEM. Representative images 6 weeks post-treatment of control limbs (F, I) and injured limbs (G, H, J, K) treated with scaffolds (G, J) or bolus injections (H, K) delivering cells and VEGF/IGF, showing direct GFP fluorescence (green) on longitudinal (F, G, H) and transverse (I, J, L) cryosections of muscle harvested 6 weeks after transplantations. Size bars correspond to 100 μm. See also Figures S1 and S2.

Figure 4. Muscle weight and size

(A) Photographs of explanted tibialis anterior muscles at 3 days following treatment with blank alginate (left) and scaffold delivering cells and VEGF/IGF-1 (right). Size bar is shown on the photomicrograph. (B) The weight of the uninjured tibialis muscles (Control) at 3 days, 2 weeks and 6 weeks, compared to muscles after myotoxin/ischemia injury and treatment with blank alginate scaffold, scaffold delivering VEGF/IGF-1, scaffold delivering cells and VEGF/IGF-1, and bolus delivery of cells and VEGF/IGF-1 in PBS. Values represent mean ± SD (n=6) in all graphs. At *p<0.05 level the means are significantly different compared with the control and the blank alginate.

Figure 5. Analysis of muscle regeneration

(A) Representative photomicrographs of tibialis muscle tissue sections from injured hindlimbs of C57 mice at post-treatment 3 days and 6 weeks, stained with H&E. Cross and longitudinal sections of injured muscles treated with blank alginate scaffold, scaffold delivering VEGF and IGF-1, scaffold delivering cells and VEGF/IGF-1, and bolus delivery of cells and VEGF/IGF-1 in PBS. (B) The number of centrally located nuclei in muscle fibers at 3 days and 6 weeks post-treatment. Values represent mean ± SD (n=6) in all graphs. ANOVA indicates statistical significance of differences between the different conditions (*p<0.05).

Figure 6. Fiber type composition analysis

(A) Fluorescent microscopy images of tibialis muscle fibers at 3 days after treatment with type I fibers (green), type II fibers (red) and type IIC fibers (yellow, colocalization of red and green). Images were taken at 10X magnification. (B) Quantification of type I and type IIC fibers at 3 days after treatment, normalized to the total cross-sectional area of each muscle. Values are mean ± SD(n=6).

Figure 7. Quantification of blood vessel densities and hindlimb perfusion

(A) Photomicrographs of tibialis muscles immunostained for the endothelial marker CD-31 at postoperative 6 wks and treatment with blank alginate, scaffold delivering VEGF/IGF-1, scaffold delivering cells and VEGF/IGF-1, bolus delivery of cells and VEGF/IGF-1 in PBS, and control (non-operated) limbs. Size bars correspond to 50 μm. (B) Quantification of blood vessel densities in tibialis muscles at 3 days and 6 weeks after myotoxin/ischemia injury and treatment with blank alginate, scaffold delivering VEGF and IGF-1, scaffold delivering cells and VEGF/IGF-1, bolus delivery of cells and VEGF/IGF-1 in PBS and, control (non-operated) limb. Values are mean ± SD (n=6). *p < 0.05 vs. blank alginate and bolus. (C) Representative color-coded laser Doppler perfusion images (LDPI) at various time points (after surgery, at 3 days, 2, 4 and 6 weeks post-operation) of mice for all conditions. (D) Quantification of LDPI for C57 mice hindlimbs treated with (black square) blank alginate, (gray circle) scaffold delivering VEGF and IGF-1, (gray triangle) scaffold delivering cells and VEGF/IGF-1 and, (black diamond) bolus delivery of cells and VEGF/IGF-1 in PBS. *p<0.05 compared to the blank alginate and bolus; mean values are presented with SD (n=6).

Figure 8. Interstitial fibrotic collagen deposition and functional recovery of skeletal muscles

(A) Representative photomicrographs of tissue sections from tibialis muscles stained to highlight the deposition of interstitial fibrotic collagen. Sections were stained with Masson’s trichromic, and conditions include tibialis muscles from uninjured hindlimbs (control) and hindlimbs of mice at postoperative 3 days, 2 weeks and 6 weeks treated with blank alginate, scaffold delivering VEGF/IGF-1, scaffold delivering cells and VEGF/IGF-1 and, bolus delivery of cells and VEGF/IGF-1 in PBS. Images are representative of 5 independent experiments. (B) Quantitative analysis of the remaining defect area 3 days and 6 weeks after injury (*p<0.05, as compared with all other conditions; **p<0.05 compared with blank scaffolds). Values represent mean and SD (n = 5). (C) Tetanic force generation of the anterior tibialis muscles of mice was measured at 3 days, 2 and 6 weeks after treatment. Tetanic force was normalized to each muscle’s weight to obtain weight-corrected specific force. Stimulation was evoked via parallel wire electrodes with 2.0 ms pulse width and 1 sec train duration, and the maximal stimulation was measured at 15V-300Hz. Mean values are presented with SD (n=6); *p<0.05 compared to the other conditions.