Generation of eX vivo-vascularized Muscle Engineered Tissue (X-MET)

Abstract

The object of this study was to develop an in vitro bioengineered three-dimensional vascularized skeletal muscle tissue, named eX-vivo Muscle Engineered Tissue (X-MET). This new tissue contains cells that exhibit the characteristics of differentiated myotubes, with organized contractile machinery, undifferentiated cells, and vascular cells capable of forming "vessel-like" networks. X-MET showed biomechanical properties comparable with that of adult skeletal muscles; thus it more closely mimics the cellular complexity typical of in vivo muscle tissue than myogenic cells cultured in standard monolayer conditions. Transplanted X-MET was able to mimic the activity of the excided EDL muscle, restoring the functionality of the damaged muscle. Our results suggest that X-MET is an ideal in vitro 3D muscle model that can be employed to repair muscle defects in vivo and to perform in vitro studies, limiting the use of live animals.

Figure 1. X-MET formation and characterization of cell populations participating to the process.

(a) Prospective isolation of progenitor populations from skeletal muscle, analysed by flow cytometer. Viable cells were identified based on forward/side scatter, SYTOX® Blue was used to exclude dying or dead cells. Haematopoietic (CD45) were also excluded from analysis. CD31⁻cells (red area) but not CD31⁺cells were positive for Sca-1 and α7 integrin. 60% of CD31⁻cells were Sca-1⁻/α7⁺ representing the myogenic compartment, while about 17% of CD31⁻cells were Sca-1⁺/α7⁻. (b) Monolayer of differentiated myotubes and committed myoblasts on a substrate of fibroblasts after 5 days in culture. (c) Initial phase of delamination. (d) Delaminated monolayer. (e) Self-organized cylindrical structure obtained 2 days after anchoring the monolayer. Scale bar, 200 μm.

Figure 2. Muscle gene expression and morphological organization.

(a) Histograms show the expression of relevant markers, measured by qRTPCR, of committed and activated satellite cells during a time course of X-MET formation. At time 0 the X-MET showed a statistically very significant up regulation of MyoD if compared to the other time points (***p < 0.001), while the expression of myogenin remained unchanged over time. (b) Immunofluorescence analysis for MyHC, MyoD and Myogenin expression on cross sections of X-MET at 10 days in culture. Scale bar, 100 μm. (c) Immunofluorescence analysis performed on cross sections of X-MET, cultured for 10 days, for laminin expression; insert shows laminin expression in skeletal muscle. Hoechst was used for nuclear staining (blue). Scale bar, 100 μm. (d) Immunofluorescence analysis performed on cross sections of X-MET, cultured for 10 days, for ERTR7 expression and MyHC. Merge image reveals the presence of myotubes positive for MyHC (red) surrounded by connective layers composed by fibroblast positive for ERTR7 (green). Hoechst was used for nuclear staining (blue). Scale bar, 10 μm. (e) Light microscopy of whole mount X-MET, after 10 days in culture. Light microscopy image revealed the presence of mononucleated cells (black arrows), localized at the typical anatomical site of satellite cells; insert shows an additional X-MET with evident satellite cells-like (black arrows). (f) Immunofluorescence analysis performed on whole mount X-MET, cultured for 10 days, for Pax-7 expression and laminin. The image reveals the presence of a satellite cell (red) surrounded by basal lamina positive for laminin (green). Scale bar, 100 μm. Insert shows a higher magnification of Pax-7 positive cell. Scale bar, 10 μm. (g) Cluster of myogenic positive cells on the X-MET surface. Scale bar, 10 μm. (h) X-gal staining of whole mount X-MET, after 10 days in culture. The presence of satellite cells was confirmed by Des/nls-LacZ transgene activation by X-gal staining. Scale bar, 100 μm.

Figure 3. Expression of endothelial markers and vessel-like structures guarantee survival of X-MET.

(a) Hematoxylin and eosin staining on cross sections of 10 days cultured X-MET revealed the presence of lumen-like structures (black arrows). Scale bar, 100 μm. (b) In vitro vascularization of engineered skeletal muscle tissue. X-MET sections were immunostained with anti-CD31 antibodies (green) showing endothelial structures throughout the 3D constructs: the cross section shows the organization of endothelial cells forming a perimeter ring with interior rays which permeate the 3D structure of X-MET, while the longitudinal section shows the presence of endothelial structures along the myotubes. Scale bar, 100 μm. (c) Immunofiuorescence analysis of X-MET and skeletal muscle cross sections for anti-CD31 (green) and smooth muscle actin (red) expression. Hoechst was used for nuclear staining (blue). X-MET merge image shows the contiguous signals of smooth muscle cells (red) and endothelial cells (green). The organization and subcellular localization of CD31 and smooth muscle actin in the X-MET is similar to that observed in skeletal muscle. Scale bar, 25 μm. (d) Relative quantitative real time PCR analysis for the expression of endothelial markers between cells cultured in monolayer (2D) and X-METs (3D) after 5 and 10 days in culture. The values represent mean ± SD of three independent experiments, each obtained pooling 5 samples together. *p < 0.05, **p < 0.01, ***p < 0.005, ^#p = 0.06 (2-way ANOVA test, Bonferroni post hoc-test). (e) Histograms show morphometric analysis. Fiber size for X-MET of 5-day (white bars), X-MET of 10-day (red bars) and X-MET of 40-day (black bars) (mean ± SEM); X-MET of 5-day = 319.5 ± 16.8 μm²; X-MET of 10-day = 303.6 ± 11.5 μm²; X-MET of 40-day = 231.0 ± 11.7 μm². The difference in the median values between the X-MET of 40-day and the other time points is statistically significant, Mann-Whitney Rank Sum test: p < 0.001.

Figure 4. Functional analysis.

(a) Graph showing spontaneous contraction of X-MET. (b) Force-frequency curves of X-MET (n = 8) compared to control skeletal muscles: EDL (n = 8) and Soleus (n = 8) muscles. Forces generated by incremental stimulation frequencies were normalized by maximal force. Eight pulse trains of frequencies ranging from 10 to 70 Hz were delivered to the X-MET to obtain the force-frequency curve; tetanic force increased with increasing frequency up to 70 Hz, but, on average, the asymptotic value is achieved at 60 Hz.

Figure 5. In vivo analysis of transplanted X-MET engineered muscle construct.

(a) Histochemical and immunohistological analysis of a transversal section of the transplanted area. Left panel: hAP stain (blue-violet coloration) shows fibers of transplanted X-MET. Middle panel: immunofluorescence analysis for embryonic myosin heavy chain (e-MyHC). hAP positive cells express also e-MyHC, revealing the donor origin of these cells. Right panel: immunofluorescence analysis for e-MyHC (red), laminin (green), and nuclei (blue). X-MET shows a histological continuity with the larger myofibers of recipient origin (at the top of panel). Scale bar, 100 μm. (b) Immunofluorescence for CD31 expression reveals the presence of endothelial structures within the area were X-MET was transplanted. Hoechst was used to stain nuclei (blue). Scale bar, 100 μm.

Figure 6. Transplanted X-MET rescue the functional properties of excised EDL muscle.

(a–b–c) Schematic illustration of the grafting. (a) The extensor digitorum longus (EDL) was exposed. (b) The ends of the X-MET were sutured on the host EDL. (c) The EDL was transacted and excised. (d–e) Functional evaluation of transplanted X-MET (n = 6). (d) The transplanted limb, in which the excised EDL muscle was replaced with X-MET, was able to catch an object (Pasteur pipette). (e) The contralateral limb was unable to do it. (f) Histograms show the results of the grip test; the X-MET transplanted mice display a 14% higher force than the peak force of the mice without X-MET (*p < 0.05, non parametric, Kruskal-Wallis ANOVA test).