Muscle tissue engineering using single fibre isolation technique: in vitro and in vivo prospectives

Introduction : Tissue engineering is a developing strategy to replace or repair congenital or acquired large muscle defects and to improve the outcome of muscle dystrophies and others genetic deficiencies. Objectives : In our work we have combined cell biology and polymer chemistry to create in vitro the most appropriate microenvironment for satellite cells in order to regenerate muscle tissue in vivo. Methods : Following this propose, we have cultured satellite cells using single muscle fiber technique to obtain a pure myogenic cellular population. Flexor digitorum brevis (FDB) muscles of adult GFP positive mice were carefully removed. The single muscle fibers, obtained after enzymatic digestion, were plated in dishes coated with matrigel. The satellite cells obtained from the fibers were expanded and seeded on to polymers. The polymers were made in PLGA (polyglycolic acid), that dissolves progressively when inserted into the muscles, and, according to the chemistry, designed to align the cells and to create a well organized system to vehicle satellite cells in vivo. We performed biomolecular analysis (PCR and western blot) and we tested muscle markers expression, with immunostaining and cytofluorimeter investigation, to affirm that our polymers are a good support, maintaining cell myogenicity. The polymers, with incorporated GFP positive cells, have been introduced in tibialis anterior muscle of isogenic wild type mice after mechanical damage. At 1, 2, 4, 8 weeks the animals have been sacrificed and serial sections of treated muscle have been analyzed with immunofluorescence Results : We observed the in vivo activation of the transplanted satellite cells that were able to form fully differentiated muscle fibers. Furthermore the transplanted cells were also able to migrate from the polymer to the damaged situ. Conclusions : This strategy could be used in the future as therapeutic tool for degenerative muscle diseases and/or for large muscle defects.