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Abstract
Several focal skeletal muscle diseases, including tumours and trauma lead to a limited loss of functional muscle tissue. There is still no suitable clinical approach for treating such defects. A promising approach could be the tissue engineering of skeletal muscle. However, a clinically reliable differentiation stimulus for three-dimensional (3-D) cultures is necessary for this process, and this condition has not yet been established. In order to quantify and analyze the differentiation potential of electrical cell stimulation, primary myoblasts were stimulated within a 3-D fibrin-matrix. Gene expression of MyoD, myogenin and AChR-ε were measured by real-time RT-PCR over a time period of eight days, showing immediate down-regulation of all marker genes. For tissue engineering approaches, cell multiplication is crucial for acquisition of sufficient tissue volumes for reconstruction. Therefore, all experiments were performed with high and low passaged myoblasts, demonstrating higher transcript rates of marker genes in low-passage cells. Our findings strongly suggest a reconsideration of electrical stimulation in muscle tissue engineering.
Keywords: skeletal muscle tissue engineering * electrical stimulation * real-time RT-PCR * 3-D cell culture * fibrin * myoblast differentiation
Introduction
Since loss of skeletal muscle tissue, due to trauma or tumour ablation e.g., often results in a scar forming healing process, the final aim of skeletal muscle tissue engineering would be the regeneration of skeletal muscle tissue. Different approaches to recreating skeletal muscle tissue in vitro and in vivo have been established [1, 2], which stand besides the attempt to recreate tissues from embryonic stem cells [3]. The latter ones still faces the obstacles of legal, moral and biological issues coming along with the use of embryonic cells. Our approach of skeletal muscle tissue engineering focuses on the cultivation and replantation of autologous muscle precursor cells, namely myoblasts. One major difficulty is based on the need of expanding cells in vitro for subsequent autologous transplantation, in order to create sufficient volumes of neo-tissue [4]. After the expansion of myoblasts, one needs to establish techniques which induce the differentiation of these cells in the direction of functional myofibers. Ideally, these techniques should be feasible in a clinical scenario, which means the absence of mutagenic substances, genetic manipulation of cells, or methods that bear long-term risks (like retro-viral transfections). Therefore, our approach to...