RESUMO
Real-time, quantitative measurement of muscle progenitor cell (myoblast) differentiation is an important tool for skeletal muscle research and identification of drugs that support skeletal muscle regeneration. While most quantitative tools rely on sacrificial approach, we developed a double fluorescent tagging approach, which allows for dynamic monitoring of myoblast differentiation through assessment of fusion index and nuclei count. Fluorescent tagging of both the cell cytoplasm and nucleus enables monitoring of cell fusion and the formation of new myotube fibers, similar to immunostaining results. This labeling approach allowed monitoring the effects of Myf5 overexpression, TNFα, and Wnt agonist on myoblast differentiation. It also enabled testing the effects of surface coating on the fusion levels of scaffold-seeded myoblasts. The double fluorescent labeling of myoblasts is a promising technique to visualize even minor changes in myogenesis of myoblasts in order to support applications such as tissue engineering and drug screening.
Assuntos
Corantes Fluorescentes/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Camundongos , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Fator Regulador Miogênico 5/genética , Fator de Crescimento Transformador alfa/metabolismo , Proteínas Wnt/agonistasRESUMO
For treatments requiring split-thickness skin grafts, it is preferable to mesh the grafts. This reduces the amount of excised skin and covers more wound area. The mesh technique, however, destroys surface continuity, which results in scarring. Strain-based bioreactors, on the other hand, have successfully expanded split-thickness skin grafts in vitro within a 7-day period, increasing graft coverage. After in vitro expansion, the expanded skin grafts were tested in a porcine full-thickness excisional wound model. Expanded graft take rate was 100%. Volumetric, histologic, and mechanical assessments indicated that expanded grafts were comparable to unexpanded grafts (positive control). While there was considerable variation in expansion (31% to -3.1%), this technique has the potential to enhance the coverage area of skin grafts while reducing or eliminating scarring.