Myofibers, the basic structural elements of skeletal muscle tissue, are formed and regenerated after injury in a unique series of events that include myoblasts adhesion, fusion and differentiation. In this process a key role is played by morphological, mechanical and biochemical stimuli provided by the extracellular environment in vivo. Traditional in vitro two-dimensional (2D) cell culture systems have been very useful to elucidate early steps of myogenesis. However, cells cultured on flat substrates differ considerably in their morphology, cell-cell/cell-matrix interaction, and differentiation from those in the physiological threedimensional (3D) environments. The aim of this work was to engineer three-dimensional (3D) human skeletal myofibers in vitro for: i) studying human myogenesis in an in vivo-like physiological microenvironment, ii) developing 3D implantable myofibers for repairing muscle defects. To achieve myoblasts spatial organization and alignment, we designed a soft hydrogel (HY) scaffold with 3D parallel micro-channels (80-160 μm in diameter, 10-15 mm long) functionalized with Matrigel. The HY ensures mass transport of metabolite and cytokines required for the proper myoblasts growth and differentiation. HY chemical composition was optimized in order to obtain a soft scaffold surrounding myobasts and myotubes, with mechanical properties (elastic modulus, E) similar to those of the physiological microenvironment of muscle in vivo (E≈12±4kPa). Human myoblasts (1÷3x104 cells/channel) were injected into the micro-channels and cultured for up to 10 days. The composition of HY was optimized based on the final application: poly-acrylamide was used for in vitro studies, while hyaluronic acid for in vivo experiments. The developed HY were biocompatible and maintained the expression of myogenic markers such as desmin. After 10 days of culture, tightly packed human myotubes bundles have been obtained, expressing the differentiation markers myosin heavy chain, α-actinin and dystrophin. It is worth to underline that we observed spontaneous contractions of human myotubes bundles. Further to be three-dimensional, thanks to their relevant dimensions (up to 15 mm in length) and their compact and elastic nature, myotubes bundles could be easily manipulated for surgical implantation. GFP+ve muscle precursors cells were cultured into the channels and implanted in the tibialis anterioris of syngenic wilde type mice. After two weeks, the HY scaffold was completed degraded, without forming fibrous tissue, and implanted cells migrated from the implantation site and gave rise to newly formed myofibers: GFP+ve with central nuclei. Taken together, the obtained results showed that the 3D HY scaffold surrounding myoblasts simulates in vitro the mechanical and biochemical properties of the physiological cell microenvironment, allowing the formation of human differentiated and contracting myotubes bundles. On the other hand, in vivo studies showed an optimal degradability of the scaffold and the formation of new myofibers integrated within the host tissue.

ENGINEERING A 3D IN VITRO MODEL OF HUMAN SKELETAL MUSCLE

SERENA, ELENA;VETRALLA, MASSIMO;GIULITTI, STEFANO;VITIELLO, LIBERO;ELVASSORE, NICOLA
2014

Abstract

Myofibers, the basic structural elements of skeletal muscle tissue, are formed and regenerated after injury in a unique series of events that include myoblasts adhesion, fusion and differentiation. In this process a key role is played by morphological, mechanical and biochemical stimuli provided by the extracellular environment in vivo. Traditional in vitro two-dimensional (2D) cell culture systems have been very useful to elucidate early steps of myogenesis. However, cells cultured on flat substrates differ considerably in their morphology, cell-cell/cell-matrix interaction, and differentiation from those in the physiological threedimensional (3D) environments. The aim of this work was to engineer three-dimensional (3D) human skeletal myofibers in vitro for: i) studying human myogenesis in an in vivo-like physiological microenvironment, ii) developing 3D implantable myofibers for repairing muscle defects. To achieve myoblasts spatial organization and alignment, we designed a soft hydrogel (HY) scaffold with 3D parallel micro-channels (80-160 μm in diameter, 10-15 mm long) functionalized with Matrigel. The HY ensures mass transport of metabolite and cytokines required for the proper myoblasts growth and differentiation. HY chemical composition was optimized in order to obtain a soft scaffold surrounding myobasts and myotubes, with mechanical properties (elastic modulus, E) similar to those of the physiological microenvironment of muscle in vivo (E≈12±4kPa). Human myoblasts (1÷3x104 cells/channel) were injected into the micro-channels and cultured for up to 10 days. The composition of HY was optimized based on the final application: poly-acrylamide was used for in vitro studies, while hyaluronic acid for in vivo experiments. The developed HY were biocompatible and maintained the expression of myogenic markers such as desmin. After 10 days of culture, tightly packed human myotubes bundles have been obtained, expressing the differentiation markers myosin heavy chain, α-actinin and dystrophin. It is worth to underline that we observed spontaneous contractions of human myotubes bundles. Further to be three-dimensional, thanks to their relevant dimensions (up to 15 mm in length) and their compact and elastic nature, myotubes bundles could be easily manipulated for surgical implantation. GFP+ve muscle precursors cells were cultured into the channels and implanted in the tibialis anterioris of syngenic wilde type mice. After two weeks, the HY scaffold was completed degraded, without forming fibrous tissue, and implanted cells migrated from the implantation site and gave rise to newly formed myofibers: GFP+ve with central nuclei. Taken together, the obtained results showed that the 3D HY scaffold surrounding myoblasts simulates in vitro the mechanical and biochemical properties of the physiological cell microenvironment, allowing the formation of human differentiated and contracting myotubes bundles. On the other hand, in vivo studies showed an optimal degradability of the scaffold and the formation of new myofibers integrated within the host tissue.
Poster abstracts of the 12th Annual Meeting of the International Society for Stem Cell Research
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3152149
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